Baculovirus-based production of biopharmaceuticals free of contaminating baculoviral virions

ABSTRACT

The present invention relates to methods for the production of biopharmaceuticals implementing a baculovirus-based system. These methods advantageously allow the production of biopharmaceuticals with a reduced number of or without contaminating baculoviral virions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 15/841,359, filedDec. 14, 2017, which is a continuation of U.S. Ser. No. 14/670,459,filed Mar. 27, 2015, now U.S. Pat. No. 9,862,934, which is acontinuation of U.S. Ser. No. 13/390,806, filed Feb. 16, 2012, now U.S.Pat. No. 8,993,317, which is the U.S. national stage application ofInternational Patent Application No. PCT/EP2010/061456, filed Aug. 5,2010, the disclosures of which are hereby incorporated by reference intheir entirety, including all figures, tables and amino acid or nucleicacid sequences.

The Sequence Listing for this application is labeled “Seq-List.txt”which was created on Feb. 9, 2012 and is 117 KB. The entire contents ofthe sequence listing is incorporated herein by reference in itsentirety.

The present invention relates to methods for the production ofbiopharmaceuticals implementing a baculovirus-based system. Thesemethods advantageously allow the production of biopharmaceuticals withreduced or no contaminating baculoviral virions.

Over the past two decades the baculovirus-insect cell technology hasbecome a very frequently used eukaryotic expression system for theproduction of recombinant proteins, not only for scientific purposes,but more and more for human and veterinary medicine (Condreay and Kost,2007, van Oers, 2006). In particular, recombinant baculoviruses derivedfrom Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV)are widely employed for large-scale production of heterologous proteinsin cultured insect cells. The main reasons for the frequent applicationof this system are: (1) high levels of expression of foreign proteins,(2) insect cells are able to grow in a suspension culture and thus areeasy to scale up, (3) the proteins synthesized in insect cells areprocessed and modified post-translationally, (4) well-developedmanipulation techniques for the viral vectors resulting in a flexibleexpression system, and 5) non-pathogenic to humans, as the baculovirushost range is restricted to insects and invertebrates. Recombinantbaculovirus vectors are being used for the production of individualproteins, as for sub-unit vaccine purposes, but also for higher orderstructures containing one or more proteins, such as enzyme complexes,viruses or virus-like particles.

Virus-like particles (VLPs) are highly organised structures thatself-assemble from virus-derived structural proteins. These stable andversatile nano-particles possess excellent adjuvant properties capableof inducing innate and acquired immune responses (Ludwig & Wagner,2007). During the past years, VLPs have been applied in other branchesof biotechnology taking advantage of their structural stability andtolerance towards manipulation to carry and display heterologousmolecules or serve as building blocks for novel nanomaterials. Forimmuno-therapeutic and prophylactic applications, many types ofvirus-like particles (VLP) have been successfully produced inbaculovirus-infected insect cells (Noad & Roy, 2003, van Oers et al.,2006, Ramqvist et al., 2007). The first commercial achievement ofbaculovirus VLP technology for use in humans is the human papillomavirus(HPV) vaccine recently marketed by GlaxoSmithKline, prophylactic againstHPV strains 16 and 18. The L1 protein of each of these types of HPV wasexpressed via a recombinant baculovirus vector and the resulting VLPswere combined to produce the vaccine Cervarix™ (Harper et al., 2006).

Today, there is a huge effort to develop baculovirus-derived influenzavirus-like particles as well as influenza subunit-vaccines as a newgeneration of non-egg and non-mammalian cell culture-based candidatevaccine. Non-replicating influenza virus-like particles are effective ineliciting a broadened, cross-clade protective immune response toproteins from emerging H5N1 influenza isolates giving rise to apotential pandemic influenza vaccine candidate for humans that can bestockpiled for use in the event of an outbreak of H5N1 influenza (Brightet al., 2008). An influenza subunit vaccine produced in insect cells isclose to FDA approval (Cox and Hollister, 2009). Similar strategiescould in principle be applied for vaccines against the pandemicinfluenza such as the recent outbreak of Swine flu.

For gene therapy purposes, baculovirus-insect cell technology is alsobeing applied for the production of infectious adeno-associated virusvectors (e.g. Urabe et al., 2002) and lentiviral vectors (Lesch et al.,2008). For the production of AAV vectors insect cells are co-infectedwith three recombinant baculoviruses—one producing the AAV replicase(REP) proteins, one carrying the cap functions for producing the AAVviral structural proteins (VP1, VP2. VP3), and a third baculoviruscomprising an AAV-ITR vector with the ability to carry and transfertransgenes. Recently an improved version of this production had beenpublished which is based on the use of only recombinant baculoviruses,one of them carrying the rep and cap functions of AAV (Smith et al.2009). The produced AAV vector is indistinguishable from that producedin mammalian cells in its physical and biological properties. The yieldof the AAV-ITR vector particles approached 5×10⁴ per Sf9 insect celldemonstrating that the system is able to produce high quantities of AAVvectors in a simple manner. Currently, clinical trials withbaculovirus-derived AAV vectors are underway for instance forlipoprotein lipase deficiency (Amsterdam Molecular Therapeutics B.V.).As an alternative, scalable approach to produce lentiviral vectors(Lesch et al., 2008) mammalian 293T cells were transduced simultaneouslyby four recombinant baculoviruses produced in insect cells to expressall elements required for generation of a safe lentivirus vector. Theunconcentrated lentiviral titers in mammalian cell culture media were onaverage 2.5×10⁶ TU ml⁻¹, comparable to titers of the lentivirusesproduced by conventional four-plasmid transfection methods. In addition,there is a general effort to convert lentiviral vector productionmethods into better scalable insect cell-based technologies.

Tjia et al., 1983 discovered that BVs can be internalized by mammaliancells and even some of the viral DNA reached the cell nucleus. Furtherstudies showed that baculoviruses can enter mammalian cells and mediateexpression of Escherichia coli chloramphenicol acetyl-transferase underthe Rous sarcoma virus promoter (Carbonell et al., 1985). These findingsled to the development of novel baculovirus-based gene delivery vehiclesfor mammalian cells (Boyce & Bucher, 1996, Hofmann et al., 1995,Condreay and Kost, 2007, Kaikkonen et al., 2008). Today, there is strongevidence that baculovirus-derived gene delivery vectors can mediatetransient and stable expression of foreign genes in mammalian cellsfollowing antibiotic selection (Lackner et al., 2008).

There is still poor knowledge about transcriptional activities ofbaculovirus promoters in mammalian cells. It has been demonstrated thatthe transactivator protein IE1 of AcMNPV is functional in mammaliancells (Murges et al., 1997) as well as the early-to-late (ETL) promoter(Liu et al., 2006a,b). Among the other imperfectly explored areas is theinteraction of baculoviruses with components of the mammalian immunesystem. AcMNPV virus is able to induce antiviral cytokine production,which protects cells from infection with vesicular stomatitis virus andinfluenza virus (Abe et al., 2003, Gronowski et al., 1999). AcMNPV isalso recognized by Toll-like receptor 9 on dendritic cells andmacrophages, and AcMNPV induces antitumor acquired immunity (Kitajima &Takaku, 2008). These results suggest that AcMNPV has the potential to bean efficient virus or tumor therapy agent which induces innate andacquired immunity. In spite of universally positive effects of AcMNPV oncomponents of the humoral and adaptive cell-mediated immunity in mice,the interaction of baculoviruses with the human immune system can beslightly different. Additionally, immunoadjuvant properties of AcMNPVshould be fully separated from immune response against targetvaccine/biopharmaceuticals produced in insect cells.

These features of baculoviruses are strongly disadvantageous in caseswhere baculoviruses are utilized for the production of vaccines or viralvectors for therapeutical purposes (e.g. AAV, lentivirus). Contaminationof the produced biopharmaceuticals with both types of baculovirusvirions—budded virions (BVs) and occlusion-derived virions (ODVs)should, therefore, be avoided. In general, the recombinant proteins canbe produced in insect cells as cytosolic, membrane-bound, orextra-cellularly secreted proteins. The latter secreted proteins arehighly “contaminated” with baculoviral BVs present in the culturemedium. It can be very difficult to separate undesirable baculovirusvirions from produced recombinant biopharmaceuticals in some productionand purification configurations. It has been shown for instance thatthese BVs can cause problems during the purification process of AAVvectors produced with baculovirus-insect cell technology (personalcommunication O. Merten, Genethon). On the other hand, there are alsoODVs, always formed inside the nuclei of infected cells, in allconventional baculovirus-insect cell expression systems, even ifocclusion bodies are not formed, due to replacement of the polyhedrinopen reading frame by a desired gene. Analogously, these virions canco-purify with intracellularly produced recombinant proteins or VLPsduring purification process.

In summary, the separation of recombinant proteins and, especially, VLPsfrom baculovirus particles, requires a lot of effort and occurs at highcosts. In addition, it results in reduced efficiency of recombinantprotein production. Therefore, the development of an improvedbaculovirus-insect cell technology allowing high expression ofheterologous proteins while eliminating baculovirus BV and ODVproduction is highly desirable, and is the topic of this patentapplication. Such a baculovirus virion-free production system wouldrepresent a significant improvement over existing systems for theproduction of all kinds of biopharmaceuticals in insect cells.

The present invention is based on the identification of efficientbaculovirus-insect cell based methods for producing biopharmaceuticalswith reduced amounts or absence of baculovirus virions.

An object of the present invention thus provides a method for theproduction of a biopharmaceutical product, comprising:

-   -   (a) infecting a biopharmaceutical-producing insect cell with at        least one baculovirus, said at least one baculovirus comprising        a genome coding for said biopharmaceutical product, and    -   (b) maintaining the biopharmaceutical-producing insect cell        under conditions such that the biopharmaceutical product is        produced,        wherein each genome of said at least one baculovirus is        deficient for at least one gene essential for proper baculovirus        virion assembly or wherein said biopharmaceutical-producing        insect cell comprises an expression control system allowing the        inactivation of at least one gene essential for proper        baculovirus virion assembly.

In an embodiment, the invention relates to the above method, whereinsaid at least one gene essential for proper baculovirus virion assemblyis made deficient in said genome by mutation, for example by way ofnucleotide substitution, insertion or deletion.

In another embodiment, the invention relates to the above method,wherein the biopharmaceutical-producing insect cell is a recombinantinsect cell comprising a construct expressing a dsRNA specific for theat least one gene essential for proper baculovirus virion assembly, thedsRNA being optionally expressed under the control of an induciblepromoter.

In a further embodiment, the invention relates to the above method,wherein the at least one baculovirus is produced before step (a) in abaculovirus-producing cell expressing a complementing copy of the atleast one gene essential for proper baculovirus virion assembly.

In yet another embodiment, the invention relates to the above method,wherein the at least one gene essential for proper baculovirus virionassembly is selected from vp80, vp39, vp1054 and p6.9.

In another embodiment, the invention relates to the above method,wherein the deficiency or inactivation of the at least one geneessential for proper baculovirus virion assembly does not affect verylate gene expression from said baculovirus in comparison to very lategene expression from the wild-type baculovirus vector.

In yet another embodiment, the invention relates to the above method,wherein the at least one baculovirus is preferably derived from AcMNPVor Bombyx mori (Bm) NPV.

In a further embodiment, the invention relates to the above method,wherein the biopharmaceutical product is a recombinant protein, arecombinant virus, a virus-derived vector, or a virus-like particle.

In another embodiment, the invention relates to the above method,wherein the biopharmaceutical product is a recombinant AAV vector.Furthermore, the invention relates to the above method, wherein thebiopharmaceutical product is a vaccine. Representative examples ofvaccines than can be produced with the method of the present inventioninclude, but are not limited to, influenza virus-like particles orinfluenza subunit vaccines, and vaccines against Human papillomavirus.

In a further embodiment, the invention relates to the above method,wherein the biopharmaceutical product is coded by at least one geneintroduced in the recombinant baculovirus genome under the control of abaculovirus promoter, preferably the p10 or polyhedrin promoter.

Another object of the invention provides the use of a baculovirus-insectcell system for the production of a biopharmaceutical product whereinthe baculovirus-insect cell system comprises abiopharmaceutical-producing insect cell infected with at least onerecombinant baculovirus, wherein:

-   -   the, or each, recombinant baculovirus comprises a recombinant        baculovirus genome that encodes the biopharmaceutical product,        or at least one component of the biopharmaceutical product, and    -   the recombinant baculovirus genome is deficient for at least one        gene essential for proper assembly of said baculovirus, or the        biopharmaceutical-producing insect cell comprises an expression        control system allowing the inactivation of the at least one        gene essential for proper baculovirus virion assembly.

Yet another object of the invention relates to a bacmid comprising abaculovirus genome, wherein said genome is deficient for a geneessential for proper baculovirus virion assembly, preferably wherein thegenome of said baculovirus is deficient for vp80, vp39, p6.9 or vp1054.In a particular aspect, said bacmid is derived from AcMNPV and islacking the vp80 ORF.

A further object of the invention relates to a recombinant AcMNPVbaculovirus vector, wherein the genome of said baculovirus is deficientfor a gene essential for proper baculovirus virion assembly, preferablywherein the genome of said baculovirus is deficient for vp80, vp39,vp1054 or p6.9. In a particular aspect, the invention relates to arecombinant AcMNPV baculovirus lacking the vp80 ORF.

The invention has also as an object an insect cell infected with theabove mentioned recombinant AcMNPV baculovirus.

Another object of the invention relates to an insect cell, comprising aconstruct expressing a dsRNA specific for a gene essential for properbaculovirus virion assembly, preferably directed against vp80, vp39,vp1054 and/or p6.9, said construct being preferably integrated in thegenome of the insect cell.

A further object of the invention relates to an insect cell comprisingan expression cassette coding for a gene essential for properbaculovirus virion assembly. In particular, the invention relates tosaid insect cell, wherein the gene coded by the expression cassette isvp80, vp39, vp1054 and/or p6.9.

Another object of the invention relates to a method for the productionof a baculovirus deficient for at least one gene essential for properbaculovirus virion assembly, comprising the step of transfecting aninsect cell comprising an expression cassette coding for a geneessential for proper baculovirus virion assembly, with a bacmidcomprising a baculoviral genome, wherein said genome is deficient for agene essential for proper baculovirus virion assembly, preferablywherein the genome of said baculovirus is deficient for vp80, vp39, p6.9and/or vp1054, wherein the gene essential for proper baculovirus virionassembly deficient in said bacmid is the gene coded by the expressioncassette comprised in said insect cell.

The present invention relates to the production of biopharmaceuticals ininsect cells by implementing a baculoviral system, but withoutcoproduction of contaminating baculovirus virions. The methods of theinvention simplify the downstream processing of biopharmaceuticalsproduced in insect cells to a large extent.

Thus, the invention relates to methods for the production of abiopharmaceutical product implementing a baculoviral system designed toavoid the production of contaminating baculoviral virions. The method ofthe present invention comprises the infection ofbiopharmaceutical-producing insect cells with at least one baculoviruscoding for said biopharmaceutical product.

Baculoviruses are enveloped DNA viruses of arthropods, two members ofwhich are well known expression vectors for producing recombinantproteins in cell cultures. Baculoviruses have circular double-strandedgenomes (80-200 kbp) which can be engineered to allow the delivery oflarge genomic content to specific cells. The viruses used as a vectorare generally Autographa californica multicapsid nucleopolyhedrovirus(AcMNPV) or Bombyx mori (Bm)NPV (Kato et al., 2010).

Baculoviruses are commonly used for the infection of insect cells forthe expression of recombinant proteins. In particular, expression ofheterologous genes in insects can be accomplished as described in forinstance U.S. Pat. No. 4,745,051; Friesen et al (1986); EP 127,839; EP155,476; Vlak et al (1988); Miller et al (1988); Carbonell et al (1988);Maeda et al (1985); Lebacq-Verheyden et al (1988); Smith et al (1985);Miyajima et al (1987); and Martin et al (1988). Numerous baculovirusstrains and variants and corresponding permissive insect host cells thatcan be used for protein production are described in Luckow et al (1988),Miller et al (1986); Maeda et al (1985) and McKenna (1989).

According to the present invention, any genome derived from abaculovirus commonly used for the recombinant expression of proteins andbiopharmaceutical products may be used. For example, the baculovirusgenome may be derived from for instance AcMNPV, BmNPV, Helicoverpaarmigera (HearNPV) or Spodoptera exigua MNPV, preferably from AcMNPV orBmNPV. In particular, the baculovirus genome may be derived from theAcMNPV clone C6 (genomic sequence: Genbank accession no. NC_001623.1—SEQID NO:1).

The terms “Biopharmaceutical”, “Biopharmaceuticals” and“Biopharmaceutical Product” are intended to define medical drugsproduced using biotechnology. As such, biopharmaceuticals may correspondto recombinantly produced drugs such as recombinant proteins, notablyrecombinant hormones or recombinant proteins for use as vaccines,viruses, for example therapeutic recombinant AAV or other viral vectorsfor use in gene therapy, as well as virus-like particles (or VLPs). Suchbiopharmaceuticals are intended to be administered to a subject in needthereof for the prophylactic or curative treatment of a diseasecondition in said subject which may be of either human or animal origin.

A biopharmaceutical product may correspond to a single chain protein orpeptide, for example in the case of a therapeutic recombinant protein,or may be a complex structure such as a virus or a virus-like particle.In the latter two cases, the components of the complex may be expressedfrom several recombinant baculoviruses, each carrying at least onecomponent of the complex structure, or from a single baculovirus whosegenome has been genetically modified by the insertion of sequencesencoding all the components of the complex. For example, for theproduction of a recombinant AAV, a system comprising three baculovirusesmay be used: a baculovirus coding for the AAV Rep proteins, abaculovirus coding the AAV Cap proteins and a baculovirus coding theAAV-ITR genome comprising a therapeutic gene between the two AAV ITRs. Asystem comprising two baculoviruses is also available now, for which theDNA sequences coding for the AAV Rep proteins and the AAV Cap proteinsare provided by one baculovirus.

In a preferred embodiment of the invention, the heterologous gene(s)encoding the biopharmaceuticals are placed under the control of abaculoviral promoter. For example, the heterologous gene(s) is (are)placed under the control of the polyhedrin or p10 promoter, or of anyother baculoviral promoter commonly used for expression in an insectcell (e.g. ie-1, p6.9, gp64 or the Orchyia pseudotsugata (Op) MNPV ie-2promoter). In a preferred embodiment of the invention, the baculoviralpromoter is selected from very late expression promoters, for examplefrom the p10 and polyhedrin promoters, preferably under the control ofthe polyhedrin promoter.

In the method of the present invention, at least one gene essential forproper baculovirus virion assembly is either absent from the genome ofthe recombinant baculovirus(es) implemented in the above describedmethod, or its expression is prevented. The inventors have shown thatthe deletion or inactivation of such genes results in the reduction, oreven the complete absence, of budded virions and/or occlusion derivedvirions, the two forms of a baculovirus.

A “gene essential for proper baculovirus virion assembly” is a genewhose deficiency or inactivation in a baculovirus-producing cellnegatively impacts the number of BVs and ODVs produced from said cell.Such a gene may be identified as provided in the herein below examples.In particular, one can use double stranded RNAs specific for aparticular baculoviral gene to assess the impact of the absence of saidparticular gene on the production of BVs and ODVs, for example bydetecting the expression of a reporter gene present in the baculoviralgenome in the cell culture, and thus determine the spreading or absenceof spreading of the baculovirus (single-infection phenotype).Alternatively baculovirus virions may be detected by the presence ofbaculoviral structural proteins or genome sequences in the culturemedium when sampling for BV production. Both virion types may bedetected by electron microscopy.

In a preferred embodiment of the invention, the gene essential forproper baculovirus virion assembly is selected from vp80, vp39, vp1054and p6.9. More preferably, the gene is selected from vp80 and vp39, saidgene being preferably vp80.

The invention provides the inactivation of genes essential for properbaculovirus virion assembly. Several strategies may be implemented forthis purpose, and in particular: the mutation, for example by deletion,of the selected gene(s) in the recombinant baculovirus genome; or thereduction of the expression of the selected gene by an expressioncontrol system provided in the biopharmaceutical-producing insect cellintended to be infected by the baculovirus. Preferably, the expressioncontrol system involves the down-regulation by RNA interference of theexpression of the protein(s) encoded by the selected gene(s).

In one embodiment of the invention, the genome of the at least onebaculovirus implemented in the method of the invention is deficient forat least one gene essential for proper baculovirus virion assembly, inparticular for a gene coding for vp80, vp39, vp1054 and/or p6.9,preferably for vp80 and/or vp39, and even more preferably for vp80. Moreparticularly, said genome is derived from AcMNPV, more particularly fromAcMNPV clone C6 genome sequence (Genbank accession no. NC_001623.1—SEQID NO: 1). Accordingly, in one aspect the invention provides the methodas defined above, wherein the baculoviral genome is an AcMNPV genome, inparticular an AcMNPV clone C6 genome, deficient for the gene coding forvp80, vp39, vp1054 and/or p6.9, preferably for vp80 and/or vp39, andeven more preferably for vp80. As is well known in the art and specifiedin Genbank accession no. NC_001623.1, these genes are positioned asfollows in AcMNPV clone C6 genome (i.e. in SEQ ID NO:1): positions89564-91639 for vp80; positions 75534-76577 for vp39 (complementarysequence); positions 45222-46319 for vp1054; positions 86712-86879 forp6.9 (complementary sequence).

It should be noted that in case the biopharmaceutical product is acomplex product comprising various subunits each encoded by differentbaculoviruses, the genomes of all the implemented recombinantbaculoviruses are deficient for the selected essential gene, so as toavoid complementation of one genome by another. In other words, whenseveral baculoviruses are used to infect the samebiopharmaceutical-producing insect cell, each of these baculoviruses aredeficient for the same gene(s) essential for proper baculovirus virionassembly.

According to the present invention, a gene may be made deficient bymutating said gene. A mutation of a gene essential for properbaculovirus virion assembly is a modification of said gene that resultsin the complete absence of a functional essential gene product.Accordingly, said mutation may result in the introduction of one orseveral stop codons in the open reading frame of the mRNA transcribedfrom the gene essential for proper baculoviral virion assembly or maycorrespond to the deletion, either total or partial, of the geneessential for proper baculovirus virion assembly. A gene essential forproper baculoviral virion assembly may be mutated by way of nucleotidesubstitution, insertion or deletion in the sequence of all or a part ofthe wild type gene (for example in the sequence provided in GenbankAccession No. NC_001623.1, for a genome derived from AcMNPV). Themutation may correspond to the complete deletion of the gene, or to onlya part of said gene. For example, one may delete at least 50%, morepreferably at least 60%, more preferably at least 70%, more preferablyat least 80% and even more preferably at least 90% of the gene essentialfor proper baculoviral virion assembly.

The mutant baculoviral genome may be produced using standard methodswell known in the art, such as site-directed mutagenesis (see, e.g.,Sambrook et al. (1989)) and Lambda red recombination (Datsenko & Wanner,2000). The gene essential for proper baculovirus virion assembly may inparticular be deleted as provided in the below examples. In summary, onecan make use of the mutant LoxP sites described by Suzuki at al. (2005),by replacing either totally or in part the gene essential for properbaculovirus virion assembly with a reporter gene flanked by mutant LoxPsites by recombination. The reporter gene (for example the gene codingfor chloramphenicol acetyl transferase (cat) is then excised byimplementing a recombination with Cre recombinase.

This embodiment is illustrated in the below examples and is detailed forbaculoviruses whose genome has been modified by deleting a 2074-bpfragment of the vp80 ORF in the AcMNPV genome. This particular genome ispart of the present invention, but is given as a non limiting example ofwhat is a mutant baculoviral genome according to the invention.

It should be noted that recombinant engineering of the baculovirusgenome may result in the insertion of several sequences like cloningsites or recombination sites (for example one remaining LoxP site afterrecombination with Cre recombinase). This is irrelevant as long as theresulting genome is made deficient for the selected gene essential forproper baculovirus virion assembly.

In this embodiment, wherein the genome of the at least one baculovirusis deficient for at least one gene essential for proper assembly ofbaculovirus virion, the production of recombinant budded baculovirusparticles needed for the initial infection of the cells producing thebio-pharmaceuticals requires the implementation of special cellsrescuing the deficient gene, i.e. these baculovirus-producing cellsexpress the selected gene. In other terms, the baculovirus-producingcell expresses a complementing copy of the at least one gene essentialfor proper baculoviral virion assembly which is deficient in thebaculovirus genome. For example, a Sf9-derived cell line constitutivelyproducing the product of the gene essential for proper assembly of thebaculovirus virion may be established. This recombinant cell line isused for production of baculovirus seed stock while conventional insectcell lines like Sf9, Sf21 or High-five cell lines can be infected withthe produced baculovirus for heterologous expression of thebiopharmaceutical product. Accordingly, the invention also relates to aninsect cell modified so as to express a gene essential for properbaculovirus assembly, said gene being mutated in a baculovirus used forthe production of biopharmaceuticals, as defined above. Such a cell lineused for the production of the mutant baculovirus vector implemented inthe method of the present invention is referred to as a“baculovirus-producing cell”. When the baculovirus genome is deficientfor a gene essential for proper baculovirus virion assembly, thebaculovirus-producing insect cell must provide and express said gene inorder to complement the deficiency and to produce an infectiousbaculovirus. In a particular embodiment, the insect cell used for theproduction of the baculovirus is modified by transfection with anexpression cassette coding for at least one gene essential for properbaculovirus virion assembly. In an embodiment, said expression cassetteis integrated in the genome of said cell. One may also use insect cellstransiently transfected with at least one plasmid comprising theexpression cassette. The term “expression cassette” denotes a constructcomprising the coding sequence of a gene of interest functionally linkedto expression control sequences. Such an expression cassette may be aplasmid comprising the ORF of a gene essential for proper baculovirusvirion assembly placed under the control of a promoter functional in theselected insect cell, and does not contain baculoviral genome sequencesother than the gene essential for proper baculovirus virion assembly tobe complemented and optionally the promoter sequence allowing theexpression of said gene (in particular, an expression cassette is not abacmid or any other baculoviral entire genome). Exemplary expressioncontrol sequences may be chosen among promoters, enhancers, insulators,etc. In one embodiment, the complementing gene is derived from thegenome of the baculovirus in which the gene essential for properbaculovirus virion assembly has been made deficient. In anotherembodiment, the complementing gene originates from the genome of adifferent baculovirus species than the baculovirus genome used for theproduction of biopharmaceuticals. For example, the baculovirus used forthe production of biopharmaceuticals may be derived from the AcMNPVgenome, and the complementing gene introduced in thebaculovirus-producing cell is derived from BmNPV or SeMNPV. Morespecifically, the baculovirus genome may be made deficient for vp80,vp39, vp1054 and/or p6.9 and the baculovirus-producing cell may comprisea copy of a gene from BmNPV or SeMNPV able to complement these genes(e.g. as provided in the examples, p6.9 is deleted in the AcMNPV genomeand the baculovirus-producing cell provides a rescuing copy of theSeMNPV p6.9 gene).

The invention thus also provides a method for the production of abaculovirus deficient for at least one gene essential for properbaculovirus virion assembly, comprising the step of transfecting aninsect cell comprising an expression cassette coding for a geneessential for proper baculovirus virion assembly, with a bacmidcomprising a baculoviral genome, wherein said genome is deficient for agene essential for proper baculovirus virion assembly, preferablywherein the genome of said baculovirus is deficient for vp80 or vp39,p6.9 and/or vp1054, wherein the gene essential for proper baculovirusvirion assembly deficient in said bacmid is the gene coded by theexpression cassette comprised in said insect cell. According to thismethod, the gene deficient in the baculoviral genome is complemented bythe gene expressed in the insect cell. The cells transfected with thebacmid are maintained in conditions such that baculovirus virions areproduced. These produced baculovirus virions, which comprise a genomewhere at least one gene essential for proper baculovirus virion assemblyis lacking, are then collected for their subsequent use for infectingbiopharmaceutical-producing insect cells for the production of thebiopharmaceutical.

In the embodiment where the genome of the baculovirus is deficient forat least one gene essential for baculovirus virion assembly, thebiopharmaceutical-producing insect cell must be unable to complement thedeficiency of said gene. Otherwise, the deficiency would be rescued bythe biopharmaceutical-producing cell and BVs and ODVs might be produced.The presence or absence of a gene essential for proper baculovirusassembly may be monitored for example by checking said cell by a PCRspecific to said gene or by detection of the protein product of thisgene (for example by western-blot with an antibody specific to said geneproduct). Cells expressing a functional product of the gene essentialfor proper baculovirus virion assembly which has been made deficient inthe genome of the implemented baculovirus intended to infect said cellmust be disregarded as bio-pharmaceutical producing cells.

In another embodiment of the invention, the expression of the geneessential for proper assembly of baculovirus virions is controlled by anexpression control system. The term “expression control system” definesa modification of the baculovirus-producing insect cell system/thebiopharmaceutical-producing cell system and/or yet another adaptation ofthe viral genome, resulting in the specific regulation of the geneessential for proper baculovirus virion assembly. This system may be aninducible expression system (for example Tet-On, Tet-Off, ecdysone-basedsystems (Dai et al., 2005) or baculovirus homologous region (hr)containing elements, such as the hr2 system described by Aslanidi et al.(2009), allowing the desired triggering or shutdown of the essentialgene, an RNA interference expressing construct or a combination ofthese.

In a particular embodiment, the expression of the gene essential forproper assembly of baculovirus is inactivated by RNA mediated silencing,or RNA interference (Salem & Maruniak, 2007, Kanginakudru et al., 2007).Preferably, an insect-cell derived cell line, in particular aSf9-derived cell line, is established by stably transforming such a cellwith a construct coding for a gene-specific double stranded RNA (dsRNA)to silence the expression of the gene essential for proper baculovirusvirion assembly. This dsRNA expressing cell line is used for theexpression of the biopharmaceutical product after infection with therecombinant baculovirus(es) carrying the gene coding for saidbiopharmaceutical product. In this embodiment, seed stock recombinantbaculovirus(es) may be produced with conventional Sf9, Sf21 or High-Fivecell lines (i.e. without the need of a complementing copy of the gene inthe cell), since in this case the baculovirus genome comprises thewild-type gene essential for proper baculovirus virion assembly.

In yet another embodiment of the invention, the gene essential forproper baculovirus virion assembly is placed under the control of aninducible promoter, allowing either the expression or repression of saidgene under controlled conditions.

In a preferred embodiment, the number of baculovirus virions produced inthe method of the present invention is reduced by at least 50% incomparison to the number of baculovirus otherwise produced by thebiopharmaceutical-producing cell using a baculovirus genome comprisingall the genes essential for proper baculovirus virion assembly. Morepreferably, the number of baculovirus virions is reduced by at least60%, at least 70%, at least 80%, at least 90% and most preferably by atleast 95% in comparison to a wild type baculovirus genome.

As discussed above, the use of insect cell/baculovirus systems for theproduction of biopharmaceuticals in the prior art is characterized bythe co-production of huge quantities of recombinant baculoviruses (andmay be over 10⁸ pfu/ml) in parallel to the biopharmaceutical product,needing carefully developed and optimized downstream processingprotocols to inactivate and eliminate this baculovirus contamination.Inactivation can be performed by the addition of a detergent stepleading to disintegration of the lipid layer of the contaminatingbaculovirus, such as used for the purification of virus-like particlesfor vaccine purposes (porcine parvovirus-VLPs (Maranga et al. (2002)) orrotavirus-VLPs (Mellado et al. (2008)) or the purification of differentserotypes of AAV (Smith et al. 2009).

Further efficient separation steps have been used: centrifugation (Wanget al. (2000); Maranga et al. (2002); Mellado et al. (2008)),microfiltration (Tellez (2005)), negative elimination of baculovirusproteins (e.g. Mellado et al. (2008)) or positive affinitychromatography (retention/capture of a biopharmaceutical—flow through ofthe contaminating proteins, such as capture of the vp7 protein ofrotavirus by Concanavalin A chromatography (Mellado et al. (2008)),capture of the immunogenic chimeric rVP2H infectious bursal diseasevirus particles by immobilized metal-ion affinity chromatography (Wanget al. (2000)) or capture of different AAV serotypes by immunoaffinitychromatography using camelid antibodies (Smith et al. 2009). Inparticular, due to the use of highly specific immunoligands, the use ofimmunoaffinity allows the complete separation of the to be purifiedbiopharmaceutical (e.g. specific AAV) from any contaminant, and in thecase of the baculovirus system, from the huge contamination bybaculovirus due to the concomitant production of baculovirus in parallelto the biopharmaceutical.

These references present very clearly the need of these differentprocess steps for inactivating and eliminating residual baculoviruscontaminants, because without these steps, the biopharmaceutical productis still considerably contaminated by various baculovirus proteins andcannot be used for clinical purposes.

The method of the present invention allows a significant reduction ofthe number of contaminating baculovirus virions, or even a completeabsence. As a consequence, a reduced number of purification steps willbe necessary for getting a biopharmaceutical for clinical purposes (oreven no purification step if no baculoviral virion is produced). Thus,the biopharmaceutical production and purification protocol is simplifiedbecause by using the method of the present invention, the need foreliminating residual baculovirus virion is greatly reduced. In case asimplified purification protocol is still to be applied, the skilledartisan may select at least one of the above identified methods andprotocols to obtain a purified biopharmaceutical product.

Preferably, the selected essential gene is a gene whose inactivationdoes not affect baculoviral very late gene expression, compared to theoriginal baculovirus vector. In the AcMNPV genome (and otheralpha-baculoviruses), the p10 and polyhedrin promoters are the very lateexpression promoters and it should be noted that in baculovirus/insectcell production systems, the heterologous gene is most commonly insertedunder the control of these very strong promoters allowing expression ofvery large amounts of recombinant proteins. The inactivation of a geneessential for proper baculovirus virion assembly, which does not affectvery late gene expression is thus preferred. The term “does not affectvery late gene expression” denotes the fact that the level ofrecombinant protein expression from very late baculovirus promotercomprised in the genome of a baculovirus modified according to theinvention is at least 70% in comparison to the levels obtained from anon-modified genome, more preferably greater than 80%, more preferablygreater than 90%. It should be mentioned that the level of expression ofa biopharmaceutical product from a very late baculoviral promoter mayeven be greater than 100% of the level obtained with the non-modifiedvector in the method of the present invention.

Among the genes tested by the inventors, the vp80 gene is particularlypreferred since its deletion does not affect very late expression, whileit totally prevents production of BVs and results in a significantreduction in the number of intracellular nucleocapsids, the precursorsof ODVs.

Very late expression may be evaluated by placing a reporter gene, forexample a gene coding for a GFP, in particular egfp, or a luciferasegene, under the control of the polyhedrin or p10 promoter in a wild typeAcMNPV vector and in a mutant AcMNPV genome from which the essentialgene has been inactivated, and by comparing the expression of theproduct of the reporter gene from both genomes. Preferably, very lateexpression from the vector with a mutated baculovirus backbone is atleast 60% of the expression level obtained with the wild type AcMNPVvector and preferably higher than 80%, more preferably higher than 90%,as measured from a reporter gene under the control of either p10 orpolyhedrin gene promoters.

The invention also relates to a method for screening baculoviral genes,the inactivation of which could be useful for producingbiopharmaceuticals without contaminating baculovirus virions in aninsect cell—baculovirus system as defined above, comprising:

a) providing a cell culture of cells containing a baculoviral genome;

b) contacting said cell culture with means for inactivating at least onetest baculoviral gene of said baculoviral genome, for example with RNAinterference; and

c) testing virion formation from said cell culture in comparison tovirion formation from a cell culture not contacted with said means;

wherein a test gene is selected as potentially useful for producingbiopharmaceuticals if its inactivation results in a reduction ofbaculoviral virion formation.

In a particular embodiment, the method for screening of the inventionfurther comprises step d) of testing very late gene expression from thecell culture contacted with said means in comparison to very late geneexpression from a cell culture not contacted with said means;

wherein a test gene is selected as potentially useful for producingbiopharmaceuticals if its inactivation results in a reduction ofbaculoviral virion formation and if it does not affect very late geneexpression from said baculoviral genome.

The invention also relates to a method for screening baculoviral genes,the inactivation of which could be useful for producingbiopharmaceuticals without contaminating baculovirus virions in aninsect cell—baculovirus system as defined above, comprising:

-   -   inactivating at least one test gene of a baculoviral genome (for        example by deletion of said test gene in said genome);    -   evaluating baculoviral very late gene expression from said        baculoviral genome as defined above;    -   determining production of baculoviral virions from cells        containing said baculoviral genome;        wherein a gene is selected as potentially useful for the        production of biopharmaceuticals if its inactivation    -   results in a reduction in the production of baculoviral virions,        and    -   does not affect very late gene expression from said baculoviral        genome, as defined above.

In a particular embodiment of the method for screening a baculoviralgene, the inactivation of which could be useful for producingbiopharmaceuticals, the inactivation of the test gene is carried outwith dsRNA specific for said test gene. In particular, the candidatebaculovirus gene can be identified by knocking down its expression byRNA interference to test its role in virion formation.

The invention will now be illustrated with the following examples, whichare provided as non limiting exemplary embodiments of the invention.

LEGENDS TO THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication, withcolor drawing(s), will be provided by the Office upon request andpayment of the necessary fee.

FIGS. 1A-1D. dsRNA-mediated gene silencing screening. Insect Sf9 cellswere seeded in 24-well tissue culture plates (2×10⁵ cells/well) in 1 mlSf-900 II SFM culture medium at 28° C. After two hours, the culturemedium was removed, and the cells were infected with recombinantbaculovirus carrying the egfp gene under control of the polyhedrinpromoter (AcMNPV-EGFP) under standard conditions.

(A) Determination of very late gene expression level using fluorescentmicroscopy. Cells were infected at MOI=10 TCID₅₀ units/cell andtransfection with gene-specific dsRNA for vp1054, vp39, vp80, dbp andec-27 was performed at 1 h post infection (p.i.). The level of very lategene expression was checked by EGFP-specific fluorescence at 48 h p.i.dsRNAs specific for egfp and cat sequences were used as RNAi controls.(B) Measurement of very late gene expression levels by animmunoblotting-based assay. The cells were infected with AcMNPV-EGFP atMOI=1 and transfection with gene-specific dsRNA was also performed at 1h p.i. The level of very late gene expression was analyzed by using arabbit anti-EGFP polyclonal antiserum at 48 h p.i. Anti-vp39 andanti-α-tubulin antibodies were used as internal controls. (C) Titrationand detection of produced budded virions in dsRNA-treated cells. Buddedvirions were harvested at 36 hours p.i., and used either for end-pointdilution assays to measure titers of infectious virions, or forPCR-based detection to check the presence of virus particles. (D)Presence of occlusion-derived virions and rod-shaped structures in vp39-and vp80-down-regulated cells. The cells were harvested 36 hours p.i.,lysed, and the cell lysates were ultracentrifuged through a cushion of40% sucrose solution (45,000 rpm for 1 hour, Beckman SW55). Pellets wereresuspended in demi-water and analyzed by negative staining electronmicroscopy. The bars represent 100 nm.

FIGS. 2A-2B. Construction of the AcMNPV vp80-null bacmid. (A) Strategyfor construction of a vp80-null bacmid containing a complete deletion ofthe AcMNPV vp80 open-reading frame via homologous recombination in E.coli. At the first step, a 2074-bp fragment encompassing the vp80 ORFwas deleted and replaced with a sequence cassette containing thechloramphenicol (cat) resistance gene flanked by modified loxP (LE andRE) sites. Subsequently, the antibiotic resistance gene (cat) waseliminated from the bacmid sequence using the Cre/loxP recombinationsystem. The promoter sequence of the p48 gene and the polyadenylationsignal of the he65 gene remained intact. Oligonucleotide pairs were usedin PCR analysis of the wild-type locus and two vp80 knock-out genotypesto confirm the deletion of the vp80 ORF and the correctinsertion/deletion of the chloramphenicol resistance gene cassette, asindicated by unilateral arrows. Their names are designated according tonucleotide sequence coordinates. Primers for cat gene cassetteamplification are named cat-F and cat-R. (B) PCR-based detection of thepresence or absence of sequence modifications in the vp80 locus in theoriginal AcMNPV bacmid (Ac-wt), Ac-vp80null(+cat), and Ac-vp80null(−cat)bacmids. The top figure confirms the vp80 gene deletion and theinsertion of the cat cassette into the vp80 locus with primer pairs90292/90889 and cat-F/cat-R. The bottom figure shows PCR-basedverification of the correct recombination processes in the vp80 locususing the 89507/91713 primer pair.

FIGS. 3A-3C. Viral replication capacity of AcMNPV-vp80 knockout andrepaired bacmid constructs using transfection-infection assays. (A)Schematic representation of expression cassettes transposed into thepolyhedrin locus. Four repair constructs were made (vp80 driven by itsnative promoter, vp80 driven by the polyhedrin promoter, N-terminallyFLAG-tagged vp80 and C-terminally FLAG-tagged vp80, both expressed fromits native promoter). The bacmid genome backbones used for transfectionassays are indicated on the left. As positive control of viralreplication the wild type AcMNPV (bMON14272) bacmid was used. TheAc-gp64null bacmid was used as negative control representing a prototypebacmid with a “single-cell infection” phenotype. (B) Time coursefluorescence microscopy showing the propagation of the infection in Sf9cells transfected with indicated bacmid constructs. Progress of viralinfection was checked by EGFP detection at indicated times posttransfection. At 120 hours p.t., the cell culture supernatants werecollected to initiate a secondary infection. (C) Secondary infectionassay. EGFP was detected at 72 hours p. i. to signal the progress ofinfection.

FIGS. 4a-4d . Growth curves of AcMNPV-vp80null repaired bacmidconstructs generated from transfection time-course assays. Sf9 cellswere transfected with 5.0 μg of DNA from each repair bacmid. (a) vp80driven by its native promoter, (b) vp80 driven by the polyhedrinpromoter, (c) N-terminally FLAG-tagged vp80, and (d) C-terminallyFLAG-tagged vp80, both expressed from the vp80 promoter. Cell culturesupernatants were harvested at the indicated time pointspost-transfection and analysed for the production of infectious buddedvirus by a TCID₅₀ end-point dilution assay. Infectivity was determinedby monitoring EGFP expression. The points indicate the averages oftiters derived from three independent transfections, and the error barsrepresent the standard deviation.

FIGS. 5A-5B. The AcMNPV-vp80null mutant is unable to produce anyinfectious/non-infectious budded virions. The Sf9 cells wereindependently transfected with 20 μg of bacmid DNA of Ac-Δvp80 (a),Ac-wt (b), Ac-Δvp80-vp80 (c), Ac-Δvp80-pH-vp80 (d), Ac-Δvp80-FLAG-vp80(e), or Ac-Δvp80-vp80-FLAG (f). Five days p.t., the buddedvirus-enriched cell culture supernatants were ultracentrifuged andbudded viruses were observed by negative staining electron microscopy(A). The bars represent 200 nm. Parallelly, harvested budded virionswere also either separated on SDS-PAGE, blotted and immuno-detectedusing anti-VP39 antibody or used for PCR-based detection to detect thepresence of viral particles (B).

FIGS. 6A-6H. The null bacmid mutant in the vp80 gene forms small numbersof nucleocapsids, and is deficient in production of occlusion-derivedvirions. The Sf9 cells transfected either with Ac-Δvp80 (A to D),Ac-Δvp80-vp80 (E, F), or Ac-wt (G, H) were fixed, stained, embedded andthin-sectioned as described in Materials and Methods. (A) Representativeoverview of Sf9 cell transfected with Ac-vp80null bacmid mutant. (B) TheAc-vp80null mutant does form lower numbers of nucleocapsids in thevirogenic stroma (C), and no occlusion-derived virions in the ring zoneof transfected cells (D). On the other hand, repair bacmid constructAc-Δvp80-vp80 fully regenerates formation of plenty of nucleocapsids inthe virogenic stroma (E), as well as normally-appearingocclusion-derived virions in the ring zone of transfected cells (F).Representative images of the virogenic stroma (G) and the ring zone (H)of cells transfected with Ac-wt bacmid. Bars represent 500 nm.Abbreviations: Nc, nucleocapsid; NM, nuclear membrane; Nu, nucleus; RZ,ring zone; Mi, mitochondrion; ODV, occlusion-derived virions; VS,virogenic stroma.

FIGS. 7A-7B. Functional complementation of the Ac-vp80null bacmid mutantusing the trans-acting vp80 gene. The Sf9 cells were transfected witheither pIZ-flag-vp80 (A) or pIZ (B) vector, and subjected toZeocin-based selection. Three weeks post-transfection, polyclonal Zeocinresistant populations of cells were seeded to a new 6-well plate andtransfected with the Ac-vp80null bacmid mutant to check complementationactivity. Virus propagation was monitored by EGFP-specific fluorescenceat 72 h and 96 h p.t. At 120 hours p.t., the cell culture supernatantswere collected to initiate a secondary infection in untreated(wild-type) Sf9 cells (right panel). EGFP was detected at 72 hours p.i.to signal the progress of infection. EGFP was detected at 120 hours p.i.to signal the progress of infection.

FIGS. 8A-8B. Construction of an AcMNPV vp39-null bacmid. (A) Strategyfor construction of a vp39-null bacmid containing a partial deletion ofthe AcMNPV vp39 open-reading frame via homologous recombination in E.coli. At the first step, an internal 498-bp fragment of the vp39 ORF wasdeleted and replaced with a sequence cassette containing thechloramphenicol (cat) resistance gene flanked by modified loxP (LE andRE) sites. Subsequently, the cat gene was eliminated from the bacmidsequence using the Cre/loxP recombination system. The promoter sequencesof the lef-4 and cg-30 genes were not affected. Arrows indicate thepositions of oligonucleotide pairs used in PCR analysis of the wild-typelocus and two vp39 knock-out genotypes to confirm the partial deletionof the vp39 ORF and the correct insertion/deletion of the cat genecassette. Primers names are designated according to the nucleotidesequence coordinates. (B) PCR-based detection of the presence or absenceof sequence modifications in the vp39 locus of Ac-wt, Ac-vp39null(+cat), and Ac-vp39null (−cat) bacmids. The figure shows the PCR-basedverification of the correct recombination processes in the vp39 locususing the 75834/76420 primer pair.

FIGS. 9A-9C. Determination of viral replication capacity of AcMNPV-vp39knockout and repaired bacmid constructs using transfection-infectionassays. (A) Schematic representation of expression cassettes, Tn7-basedtransposed into the polyhedrin locus. (1) vp39 expressed from thepolyhedrin promoter, (2) a double gene vp39 and lef-4, both driven bytheir native promoters, (3) a double gene vp39 and cg-30 both driven bythe polyhedrin promoter, and finally (4) a double gene construct ofN-terminally FLAG-tagged vp39 driven by the polyhedrin promoter and thecg-30 ORF expressed from both its native and also the more upstreampolyhedrin promoter. The parental bacmid genome backbones used fortransfection assays are indicated on the left. The wild type AcMNPV(bMON14272) bacmid was used as positive control of viral replication.(B) Time course fluorescence microscopy showing the propagation of theinfection in Sf9 cells transfected with indicated bacmid constructs.Viral progressions were checked by EGFP detection at indicated timespost transfection. At 168 hours p.t., the cell culture supernatants werecollected to initiate a secondary infection. (C) Secondary infectionassay. EGFP detection was performed at 72 hours p.i. to measure progressof the infection.

FIGS. 10A-10B. Construction of an AcMNPV vp1054-null bacmid. (A)Strategy for the construction of a vp1054-null bacmid containing adeletion of the AcMNPV vp1054 open-reading frame via homologousrecombination in E. coli. A 955-bp sequence from the 3′-end of thevp1054 ORF was deleted and replaced with a cat sequence cassette flankedby modified loxP (LE and RE) sites. At the same time, a single pointmutation was introduced to change the first translation codon ATG→Met toACG→Thr, to prevent translation into a C-truncated VP1054 protein. Italso meant that the internal AAT codon no. 32 of lef-10 was mutated toAAC, both encoding Asn. Subsequently, the cat gene was eliminated usingthe Cre/loxP recombination system. The promoter sequence ofvp1054/lef-10 was not affected in the bacmid construct. Since thepolyadenylation signal of the lef-10 gene was removed, a novel syntheticpoly-A signal combined with stop codon (TAATAAA) was introduced at the3′-end of the lef-10 ORF. Arrows represent locations of oligonucleotidepairs used in the PCR analysis of the wild-type locus and two vp1054knock-out genotypes to confirm the deletion of the vp1054 ORF andcorrect insertion/deletion of the cat cassette. (B) PCR-based detectionof the presence or absence of sequence modifications in the vp1054 locusof Ac-wt, Ac-vp1054null (+cat), and Ac-vp1054null (−cat) bacmids. Thetop figure is showing confirmation of the vp1054 gene deletion andinsertion of the cat cassette into vp1054 locus using primer pairs90292/90889 and cat-F/cat-R. The bottom figure shows CR-basedverification of the correct recombination processes in the vp1054 locususing the 89507/91713 primer pair.

FIGS. 11A-11C. Viral replication capacity of AcMNPV-vp1054 knockout andrepaired bacmid constructs using transfection-infection assays. (A)Schematic representation of expression cassettes transposed into thepolyhedrin locus. The bacmid genome backbones used for transfectionassays are indicated on the left. Two Ac-vp1054null-derived constructswere made: first construct carrying only egfp marker gene under controlof p10 promoter, and second construct carrying both egfp marker andoverlapping lef-10/vp1054 locus directed from their natural promotersequences (d). As positive control of viral replication the wild typeAcMNPV (bMON14272) bacmid was used (a). The Ac-gp64null bacmid was usedas negative control representing a prototype bacmid with a “single-cellinfection” phenotype (b). (B) Time course fluorescence microscopyshowing the propagation of the infection in Sf9 cells transfected withindicated bacmid constructs. Progress of viral infection was checked byEGFP detection at indicated times post transfection. At 120 hours p.t.,the cell culture supernatants were collected to initiate a secondaryinfection. (C) Secondary infection assay. EGFP was detected at 72 hoursp.i. to signal the progress of infection.

FIGS. 12A-12B. Construction of an AcMNPV p6.9-null bacmid. (A) Strategyfor construction of a p6.9-null bacmid containing a complete deletion ofthe AcMNPV p6.9 open-reading frame via homologous recombination in E.coli. A 164-bp fragment of the p6.9 ORF was deleted and replaced with acat resistance gene flanked by modified loxP (LE and RE) sites.Subsequently, the cat gene was eliminated from the bacmid sequence usingCre/loxP recombination. The promoter sequence of p6.9 gene was leftunaffected, since its sequence is overlapping with the p40 ORF. Arrowsrepresent locations of primer pairs used in the PCR analysis of thewild-type locus and two p6.9 knock-out genotypes. (B) PCR-baseddetection of the presence or absence of sequence modifications in thep6.9 locus of Ac-wt, Ac-vp6.9null (+cat), and Ac-vp6.9null (−cat)bacmids. The top figure shows the insertion of the cat cassette into thep6.9 locus using primer pairs cat-F/cat-R. The bottom figure showsPCR-based verification of the correct recombination processes in thep6.9 locus using the 86596/86995 primer pair.

FIGS. 13A-130. Viral replication capacity of AcMNPV-p6.9 knockout andrepaired bacmid constructs using transfection-infection assays. (A)Schematic representation of expression cassettes transposed into thepolyhedrin locus. Two repair constructs were made (AcMNPV p6.9 andSeMNPV p6.9 genes, both driven by the AcMNPV p6.9 promoter). The bacmidgenome backbones used for transfection assays are indicated on the left.As positive control of viral replication the wild type AcMNPV(bMON14272) bacmid was used. The Ac-gp64null bacmid was used as negativecontrol representing a prototype bacmid with a “single-cell infection”phenotype. (B) Time course fluorescence microscopy showing thepropagation of the infection in Sf9 cells transfected with indicatedbacmid constructs. Progress of viral infection was checked by EGFPdetection at indicated times post transfection. At 120 hours p.t., thecell culture supernatants were collected to initiate a secondaryinfection. (C) Secondary infection assay. EGFP was detected at 72 hoursp.i. to signal the progress of infection. (D) Comparisons of growthcurves of AcMNPV-p6.9null (a), AcMNPV-p6.9null rescued with AcMNPV p6.9(b), and AcMNPV-p6.9null rescued with SeMNPV p6.9 (c) constructs withwild-type (Ac-wt) bacmid. Sf9 cells were transfected with 5.0 μg of DNAfrom each bacmid, cell culture supernatants were harvested at theindicated time points post-transfection and analysed for the productionof infectious budded virus by a TCID₅₀ end-point dilution assay.Infectivity was determined by monitoring EGFP expression. The pointsindicate the averages of titers derived from three independenttransfections, and the error bars represent the standard deviation.

FIGS. 14A-14B: Western blot analysis of Flag:vp80 in cells, BV and ODV.(A) Time course of vp80 expression in infected insect cells. Sf9 cellswere infected with the Ac-Δvp80-Flag.vp80 repair virus, and harvested atindicated time points. Flag.VP80 was detectable by western blot analysisfrom 12 h to 72 h p.i. as a band of approximately 95 kDa. In addition, asecond Flag.VP80-specific band of ˜80 kDa accumulated from 48 h until 72h p.i. Tubulin was used as an internal loading control. (B) The VP80associates with the nucleocapsid fraction of BV. Two days p.i., BVs werepurified by isokinetic ultracentrifugation in a sucrose gradient andseparated into nucleocapsid (Nc) and envelope (Env) fractions byNonidet-P40-based extraction. Flag.VP80 was detected in the Nc fractionas a double-band with molecular weights between the two variants (80-kDaand 95-kDa) detected in infected Sf9 cells (upper panel). Correctseparation into Nc and Env fractions was controlled by anti-VP39 andanti-GP64 antibodies (bottom panels). (C) VP80 is also a structuralcomponent of ODV-nucleocapsids. Sf9 cells were co-infected withAc-Δvp80-Flag.vp80 (MOI=25) and AcMNPV strain E2 (MOI=5) viruses. Fivedays p.i., ODVs were released from occlusion bodies and subsequentlyseparated into nucleocapsid (Nc) and envelope (Env) fractions. Westernblot analysis showed that VP80 is present in the DV Nc fraction as asingle band of ˜80 kDa. Proper fractionation into Nc and Env fractionswas controlled using anti-PIF-1 antiserum (bottom panel).

FIGS. 15A-15C. Functional complementation of the Ac-vp80null bacmiddefective in BV production by trans-complementation. (A) Detection ofFLAG:VP80 in a transgenic Sf9-derived cell line (Sf9-vp80) by Westernanalysis. Tubulin was used as an internal loading control. (B)Time-course fluorescence microscopy (EGFP) to follow the infection inSf9-vp80 cells transfected (i) or infected (ii) with the Ac-Δvp80 bacmid(a,b). At 120 h p.t., the cell culture supernatants were collected toinitiate a secondary infection in either Sf9-vp80 (a) or Sf9 (b) cells(panels on the right side). As negative control Ac-Δvp80 was propagatedin Sf9 cells (c), Ac-wt propagated in Sf9 cells (d) was used as positivecontrol. (C) Comparative release of infectious BV virions. Sf9-vp80cells were transfected with the Ac-Δvp80 bacmid and Sf9 cells witheither the Ac-Δvp80 (negative control) or the Ac-wt (positive control)bacmid. BVs were quantified in cell culture supernatants at 6 days p.t.by end point dilution. Representative results of three independentassays with error bars giving the SD are shown.

FIGS. 16A-16C. Analysis of foreign gene expression bytrans-complemented, replication-deficient baculovirus seed. Sf9 cellswere infected with Ac-wt, Ac-Δvp80-Flag:vp80 or Ac-Δvp80 virus seed(MOI=10 TCID₅₀ units per cell), all expressing egfp from the very latep10 promoter. (A) At 48 h p.i. the presence of EGFP, Flag:VP80 and GP64was analyzed by Western blotting. Actin was used as an internal loadingcontrol. (B) Photomicrographs of cells expressing EGFP 72 h p.i. (top),and relative amount of EGFP measured by ELISA at 48 and 72 h p.i.(bottom) (C) Photomicrographs of cells expressing EGFP 72 h p.i. (top),and analysis of BV released to test for revertant genotypes by TCID₅₀titration (bottom). The results of three independent assays are shownwith error bars (SD) (B and C).

FIGS. 17A-17D. The novel baculovirus-insect cell technology approachdesignated for the production of biopharmaceuticals free ofcontaminating baculoviral virions. (A) Insect cell engineering toexpress an essential viral factor (vp80) to complement a vp80 mutationin the virus. The transgenic Sf9 cells encode the vp80 ORF and aresistance gene allowing antibiotics-based selection of the transgeniccells. (B) Generation of an Ac-Δvp80 bacmid defective in production ofBV and ODV virions. The bacmid lacks the entire vp80 ORF. (C) Productionof a baculovirus seed stock by trans-complementation in the engineeredSf-vp80 cells. The Sf9-vp80 cells are transfected with the Ac-Δvp80bacmid to produce trans-complemented virus progeny. After budded viruspropagation, high-titer virus stocks are produced in the Sf9-vp80packaging cells. (D) Baculovirus-based recombinant protein expression.Conventional Sf9 cells are infected with the trans-complemented buddedvirus progeny. Recombinant protein is expressed from very latebaculovirus promoters (p10 or polh) allowing high levels of expression,while no contaminating baculovirus virions (BV/ODV) are produced.

EXAMPLES Example I Materials and Methods Insect Cells and Viruses

Spodoptera frugiperda (Sf9) cells were maintained in SF900-II serum-freemedium (Invitrogen) under standard conditions. Recombinantbacmid-derived AcMNPV virus (AcMNPV-EGFP) carrying an egfp reporter geneunder control of the very late polyhedrin promoter transposed into thepolyhedrin locus was obtained from Pijlman et al. (2006). The virus waspropagated and its titers were determined by an end-point dilution assayin Sf9 cells.

In Vitro Synthesis of dsRNA

The method used to synthesize dsRNA is similar to that described byRamadan et al. (2007) with minor modifications. All DNA templates werePCR amplified using primers with twenty-five nucleotide overhangshomologous to the T7 RNA polymerase promoter sequence5′-gcttctaatacgactcactataggg-3′. The sequences of the primers indicatedbelow are given in Table 1. The following primers were used foramplifying these genes: primers vp39-F and vp39-R for vp39; primers45510 and 46235 for vp1054, primers 90292 and 90889 for vp80; primersec-27-F and ec-27-R for odv-ec27; and primers dbp-F and dbp- for dbp. Totest the efficiency of the RNAi studies we made dsRNA against egfp withprimers gfp-F and gfp-R, and to have a negative control we made dsRNAwith primers cat-F and cat-R for the chloramphenicol acetyl transferase(cat) gene.

The PCR products were purified using the Illustra GFX PCR DNA and GelBand Purification Kit (GE Healthcare, Buckinghamshire, UK) and were usedas templates for dsRNA in vitro synthesis using the T7 RiboMAX™ ExpressRNAi System (Promega, Madison, Wis., USA) according to manufacturer'sprotocol. Briefly, approximately 1 μg of purified DNA templates wereused for RNA synthesis at 37° C. for 4 h. After synthesis, DNA templateswere removed by digestion with DNase. Complementary RNA strands wereannealed by incubation at 70° C. for 10 min followed by slow cooling toroom temperature (˜30 min). Non-annealed (single-stranded) RNA moleculeswere degraded by RNase A treatment (30 min, 37° C.). Finally, the dsRNAwas isopropanol precipitated, resuspended in DEPC-treated sterile waterto a final concentration of 0.5-1 mg/ml, and its purity and integritywere checked by agarose gel electrophoresis. The dsRNA was kept at −80°C. in aliquots of 40 μl. Immediately before transfection, the dsRNA wasthawed on ice.

RNAi Procedure in Baculovirus-Infected Insect Cells

Sf9 cells were seeded in 24-well tissue culture plates (2×10⁵cells/well) in 1 ml Sf900-II culture medium without serum at 28° C.After two hours, the culture medium was removed, and the cells wereinfected with recombinant baculovirus AcMNPV-EGFP at a multiplicity ofinfection (MOI) of 10 TCID₅₀ units/cell for 1 h, under standardconditions. One hour post infection (p.i.), dsRNA (20 μg/well) wasintroduced into the cells by Cellfectin™-based (Invitrogen) transfectionin Grace's serum-free medium. After 4 h, the transfection mixture wasreplaced with Sf900-II serum-free medium. The cells were incubated for atotal of 48 h p.i. at 28° C. and then harvested by centrifuging at1000×g for 5 min for Western blot and electron microscopy analysis.However, one fifth of the culture medium was harvested at 36 h p.i., andused for titration of budded virions by end-point dilution assays or forPCR-based detection of viral DNA. In all the experiments, dsRNAcorresponding to the cat gene was taken as negative control. On theother hand, egfp gene-specific dsRNA was used as positive control forthe RNAi procedure.

SDS-Polyacrylamide Electrophoresis and Western Blotting

For immuno-detection, the Sf9 cells were disrupted in 125 mM Tris-HCl,2% sodium dodecyl sulfate (SDS), 5% 2-mercapthoethanol, 10% glycerol,0.001% bromophenol blue, pH 6.8 at 95° C. for 10 min. Proteins wereseparated in 10% SDS-polyacrylamide gels, and subsequently transferredto Immobilon-P membranes (Millipore) by semi-dry electroblotting.Membranes were blocked for 30 min in 1×PBS containing 2% fat-extractedmilk powder, followed by incubation for 1 h at room temperature witheither rabbit polyclonal anti-GFP antiserum (Molecular Probes), rabbitpolyclonal anti-VP39 antiserum, or monoclonal anti-α-tubulin antibody(Sigma-Aldrich), all diluted 1/2000 in 1×PBS containing 0.2% milk power.After washing (3×10 min) in 1×PBS, the membranes were incubated with1/4000 dilution of either goat anti-rabbit IgG or rabbit anti-mouse IgGantibodies conjugated with alkaline phosphatase (Sigma). After finalwashing (3×10 min) in AP buffer (100 mM Tris-Cl [pH 9.5], 100 mM NaCl, 5mM MgCl₂), the blots were developed with 5-bromo-4-chloro-3-indolylphosphate nitroblue tetrazolium(NBT)/5-bromo-4-chloro-3-indolylphosphate (BCIP) (Bio-Rad) according tothe manufacturer's instructions.

Preparation of Viral Genomic DNA and its PCR-Based Detection

Two-hundred microliters of cell culture medium were collected at 36 hp.i. and used for preparation of viral DNA. The cells and cell debriswere removed from samples by centrifuging at 1000×g for 5 min.Supernatants containing budded virions were quantitatively transferredto new sterile tubes and centrifuged again at 12000×g for 90 min.Pelleted BVs were re-suspended in 200 μl TE buffer (10 mM Tris-HCl [pH7.5], 1 mM EDTA) containing Proteinase K (540 μg/ml), and incubated at55° C. for 2 h. A phenol:chloroform:isoamyl alcohol (25:24:1) and achloroform extraction were subsequently performed. The DNA wasprecipitated by adding an equal amount of isopropanol and the pellet waswashed with 70% ethanol. The DNA pellet was dissolved in 15 μl sterilewater, and 2 μl of the final DNA solution was applied to PCR-baseddetection of the vp39 gene sequence using primers mentioned above. AllPCR reactions were performed in 25 μl volumes including: 2 μl DNA, 200μM dNTPs, 10 pmol of each primer, 1.5 mM MgCl₂ and 1.5 U GoTaq DNApolymerase (Promega). Amplification conditions were as follows: aninitial denaturation at 94° C. for 2 min, after which 30 cycles ofdenaturation (30 s at 94° C.), primer annealing (20 s at 60° C.) andprimer extension (25 s at 72° C.). The termination cycle was 7 min at72° C. Negative controls were included in all PCR amplifications to testfor contaminants in the reagents. Aliquots (3.0 μl) of the PCR productswere analysed by electrophoresis in 1.2% (w:v) agarose gels, with 1×TAEbuffer, stained with ethidium bromide (0.5 μg/ml).

Generation of an Antibiotic Resistance Gene-Free AcMNPV Vp80-Null Bacmid

To determine whether the VP80 protein has an essential role in thecontext of viral progeny production, we constructed an AcMNPV bacmid(derived from bMON14272 (from Invitrogene)) with a deletion of the vp80ORF by homologous recombination in E. coli. To accomplish this, a catgene flanked by mutant LoxP sites (Suzuki et al., 2005) was amplifiedusing PCR primers vp80-KO-F and vp80-KO-R (see Table 1) from a plasmidcomprising a cat gene flanked by mutant LoxP sites. The resulting PCRfragment, which contained the cat gene flanked by mutant LoxP sites andAcMNPV ˜50-bp homology sequences to the 5′ or 3′ proximal region of thevp80 ORF, was treated with DpnI and gel-purified to eliminate thetemplate plasmid. The PCR product was then transformed into DH10ß E.coli cells containing bMON14272 (Invitrogen) and the Lambda REDrecombinase-producing plasmid pKD46 (Datsenko & Wanner, 2000), which hadbeen prepared in the following manner. Transformed DH10ß-bMON14272/pKD46E. coli cells were grown in 50-ml LB (2.0% peptone, 0.5% yeast extract,85.5 mM NaCl, [pH 7.0]) cultures with kanamycin (50 μg/ml), ampicillin(100 μg/ml) and L-arabinose (1.5 mg/ml) at 30° C. to an OD₆₀₀ of ≈0.6and then made electrocompetent by a standard procedure. Theelectroporated cells were incubated at 37° C. for 3 h in 3 ml LB mediumand plated on LB-agar containing chloramphenicol at a concentration of6.5 μg/ml. After 48-h incubation at 37° C., thechloramphenicol-resistant colonies were streaked to fresh LB-agar mediumwith 34 μg/ml chloramphenicol. The plates were incubated at 37° C.overnight, and colonies resistant to chloramphenicol were selected forfurther confirmation of the relevant genotype by PCR. Primers 90292 and90889 were used to confirm the absence of the vp80 ORF, and primerscat-F and cat-R were employed to verify the presence of cat cassetteinto bacmid (detailed sequences in Table 1).

To eliminate the introduced antibiotic resistance gene (cat) from thebacmid backbone, a Cre/LoxP recombinase system was employed. A Crerecombinase-carrying plasmid pCRE obtained from Jeanine Louwerse (LUMC0, The Netherlands) was introduced into DH10b-bMON14272-vp80null E. colicells, and CRE expression was subsequently induced by the addition ofisopropyl thiogalactoside (IPTG). Briefly, the electroporated cells wereincubated at 37° C. for 3 h in 3 ml of LB medium (2.0% peptone, 0.5%yeast extract, 85.5 mM NaCl, [pH 7.0]) and plated on LB-agar mediumcontaining 50 μg/ml kanamycin, 100 μg/ml ampicillin and 2 mM IPTG. After24-h incubation, colonies resistant to kanamycin and ampicillin wereselected for further verification of the desired genotype by PCR. InPCR-based analysis, primers 89507 and 91713 (Table 1) were used toverify elimination of cat gene from bacmid backbone. Positive cloneswere also confirmed by DNA-sequencing.

To recover transposition competence, the helper transposase-encodingplasmid pMON7124 (Invitrogen) was re-introduced intoDH10ß-bMON14272-vp80null E. coli cells. Finally, the egfp reporter genewas introduced into the vp80-null bacmid to facilitate observation ofits behaviour in insect cells. Briefly, the egfp reporter gene wasamplified using PCR oligonucleotides gfp-NheI-F and gfp-SphI-R (Table 1)from plasmid pEGFP-N3 (Clontech). The PCR product was cloned intoplasmid pJet1.2/Blunt using CloneJET™ PCR Cloning Kit (Fermentas)according to manufacturer's protocol. Subsequently, the egfp ORF wasexcised from error-free pJet1.2-egfp with NheI and SphI and subclonedinto NheI/SphI-digested pFastBacDUAL (Invitrogen), to generate plasmidpFB-egfp. An expression cassette containing the egfp reporter gene undertranscriptional control of the very late p10 promoter was transposedfrom pFB-egfp into polyhedrin locus of vp80-null bacmid as described inthe Bac-to-Bac manual (Invitrogen). In the resulting genome, thecomplete vp80 ORF has been removed (see FIG. 2). This corresponds to thedeletion of 2074 bp from nucleotide positions 89564 to 91637 in theAcMNPV clone C6 genome provided in SEQ ID NO: 1.

Construction of Repaired Vp80-Null Bacmids

To prepare vp80 repair donor vectors, we modified plasmid pFB-egfp(noted above) by removing the polyhedrin promoter and replacing it witha fragment containing the vp80 promoter region and the vp80 ORF. First,a 2300-bp fragment containing both the vp80 promoter and ORF sequencewas amplified using primers pvp80-StuI-F and vp80-XbaI-R (Table 1) frombacmid bMON14272 template, and cloned into vector pJet1.2/Blunt(Fermentas) to form pJet1.2-pvp80-vp80. After DNA sequence verification,the vp80 cassette was excised from pJet1.2-pvp80-vp80 by StuI/XbaIdouble digestion, and then subcloned into Bst1107I/XbaI-digested andgel-purified pFB-egfp to generate donor plasmid pFB-egfp-pvp80-vp80.Parallelly, a donor plasmid pFB-egfp-polh-vp80, where vp80 ORF is drivenby the very late polyhedrin promoter (polh) was constructed. To thisaim, a 2105-bp fragment carrying the vp80 ORF was amplified usingprimers vp80-SacI-F and vp80-XbaI-R (Table 1) and cloned intopJet1.2/Blunt, to generate pJet1.2-vp80. In the final step, the vp80 ORFwas cut out (SacI/XbaI) from pJet1.2-vp80, and subcloned intoSacI/XbaI-digested pFB-egfp, to create pFB-egfp-poIH-vp80.

To overcome a problem associated with the unavailability of anti-VP80antibody, FLAG tag decoration (N- and C-terminus fusion) of VP80 wasperformed to facilitate immunodetection. The N-terminally fusedFLAG-vp80 sequence was generated by a double-step PCR strategy, aso-called fusion PCR. First, a 259-bp fragment containing the vp80promoter and the FLAG tag was PCR amplified using primers pvp80-StuI-Fand vp80-FLAG-R1 from the bMON14272 bacmid template. Aftergel-purification and DNA quantification, the 259-bp fragment was used asforward primer in a second step PCR amplification with the reverseprimer vp80-XbaI-R on the bMON14272 bacmid template. The final PCRproduct (2324 bp) was cloned into vector pJet1.2/Blunt (Fermentas) toform pJet1.2-pvp80-FLAG-vp80. After DNA sequence verification, theFLAG-vp80 cassette was excised from pJet1.2-pvp80-FLAG-vp80 by StuI/XbaIdouble digestion, and then subcloned into Bst1107I/XbaI-digested andgel-purified pFB-egfp to generate donor plasmidpFB-egfp-pvp80-FLAG-vp80. The C-terminally fused vp80-FLAG cassette wasamplified using pvp80-StuI-F and vp80-FLAG-R from the bMON14272 bacmidtemplate. The 2324-bp fragment was cloned into pJet1.2/Blunt, andsubsequently transferred into pFB-egfp in a similar way as previousconstructs.

The inserts of all developed donor plasmids were transposed into thevp80-null bacmid following the Bac-to-Bac protocol (Invitrogen).Screening of transposition-positive constructs into the polh locus wasdone by a triplex PCR-based assay employing a M13 forward and reverseprimers and a gentamicin resistance gene-specific primer GenR (Table 1).

Transfection-Infection Assay

Bacmid DNAs were prepared from 1.5-ml over-night bacterial cultures of 2to 3 independent colonies carrying the bacmid with the insertedheterologous gene according to the Bac-to-Bac manual (Invitrogen) andwere analyzed in parallel. For transfections, 1 μg of each bacmid DNApreparation was used to transfect 1×10⁶ Sf9 cells in a 6-well plate bythe Cellfectin™-based transfection protocol as described in theBac-to-Bac (Invitrogen) manual. From 72 h to 120 h post transfection(p.t.), viral propagation was checked by fluorescence microscopy. At 120h p.t., the cell culture medium was centrifuged for 5 min at 2000×g toremove cell debris, and this clarified supernatant was used to infect1.5×10⁶ Sf9 cells in 6-well plates. After 72 h p.i., the spread of virusinfection was again monitored by fluorescence microscopy. In allexperiments, a wild-type bMON14272 bacmid carrying the egfp reportergene under control of the p10 promoter was used as positive control. AbMON14272-gp64null bacmid also carrying the egfp reporter gene undercontrol of the p10 promoter served as negative control, since it haslost the ability of cell-to-cell movement of the infection (Lung et al.,2002).

Time-Course Characterization of Viral Propagation in Cell Culture

Time course analyses were performed to compare budded virus productionof the AcMNPV-vp80null virus and the various repair constructs incomparison to the wild type AcMNPV bacmid (Ac-wt) all containing egfp.Briefly, the Sf9 cells were seeded in 6-well tissue culture plates(1×10⁶ cells/well in 1 ml Sf900-II culture medium without serum at 28°C.). After two hours, the culture medium was removed, and the cells weretransfected with 5 μg bacmid DNA, under standard conditions asrecommended in the Bac-to-Bac manual (Invitrogen). Cell culturesupernatants were harvested at 24, 48, 72, 96 and 120 h p.t., andanalysed for the production of infectious budded virus by an end-pointdilution assay to determine the tissue culture infective dose 50(TCID₅₀). Infection was determined by monitoring egfp expression (fromthe p10 promoter). The average values of infectious titers derived fromthree independent transfections were calculated and plotted into graphs.

Transmission Electron Microscopy

Insect Sf9 cells were seeded in 25T flask (3.5×10⁶ cells/flask), andtransfected with 20 μg either the Ac-Δvp80, rescue Ac-Δvp80-vp80 orAc-wt bacmid construct. After 48 h p.t., the cells were harvested andprepared for transmission electron microscopy as described previously(van Lent et al., 1990). Samples were examined and photographed with aPhilips CM12 electron microscope.

Budded Virus Production Assay

Insect Sf9 cells were seeded in two 25T flasks (3.5×10⁶ cells/flask),and transfected with 20 μg either Ac-Δvp80, Ac-Δvp80-vp80,Ac-Δvp80-pH-vp80, Ac-Δvp80-FLAG-vp80, Ac-Δvp80-vp80-FLAG, or Ac-wtbacmid construct. Five days p.t., the BV-enriched cell culturesupernatants were harvested, and ultracentrifuged through a cushion of10% sucrose solution (25,000 rpm for 1.5 hour, Beckman SW32). Pelletedbudded virions were resuspended in sterile demi-water, and prepared foreither negative staining electron microscopy, SDS-polyacrylamideelectrophoresis, or PCR-based detection (as mentioned above).

Purification of ODVs and Rod-Shaped Structures from Infected Cells

The presence of ODVs and rod-like structures in infected/transfectedinsect cells was analyzed by electron microscopy (EM). For this purpose,insect cells were harvested 48 h p.i., lysed and the cell lysates wereultracentrifuged through a 40% sucrose cushion in TE (1 mM Tris-HCl pH7.4, 0.1 mM EDTA) buffer (45,000 rpm for 1 hour, Beckman SW55). Pelletswere resuspended in sterile demi-water and analyzed by negative stainingEM as described previously (van Lent et al., 1990).

Development of Transgenic Sf9-Derived Cell Line Expressing Vp80

To develop a cell line, which produces the VP80 protein, a 2105-bpfragment carrying the vp80 ORF was amplified using primers vp80-SacI-Fand vp80-XbaI-R (Table 1) and cloned into pJet1.2/Blunt, to generatepJet1.2-vp80. In the next step, the vp80 ORF was cut out (SacI/XbaI)from pJet1.2-vp80, and subcloned into SacI/XbaI-digested pIZ(Invitrogen), to create pIZ-vp80. The resulting plasmid vector pIZ-vp80was linearized with Eco57I, and gel-purified. Sf9 cells were seeded in asix-well plate (1×10⁶ cells/well), and transfected with 10 μg of thelinearized vector. After 24 hours post-transfection, cells were selectedby cell culture medium containing Zeocin™ (300 μg/ml) for 2 to 3 weeks,until no control Sf9 cells survived under the same conditions. Cellswere then propagated as an uncloned cell line.

Generation and Characterization of a AcMNPV Vp39-Null Bacmid

To study the role of the vp39 gene in the context of viral progenyproduction and the nucleocapsid assembly process, we constructed anAcMNPV bacmid (bMON14272) with a deletion of vp39 by homologousrecombination in E. coli according to the same procedure as noted abovefor the AcMNPV vp80null bacmid construct. Since the sequence of the vp39ORF is overlapping with promoter sequences of both flanking ORFs (cg-30and lef-4), only an internal part of the vp39 ORF could be deleted, toavoid de-regulations of cg-30 and lef-4 expression. To reach this, a catgene flanked by mutant LoxP sites was amplified using PCR primersvp39-KO- and vp39-KO-R (Table 1) from a plasmid comprising a cat geneflanked by mutant LoxP sites. The resulting PCR fragment, whichcontained the cat gene flanked by mutant LoxP sites and ˜50-bp sequenceshomologous to an internal region of the vp39 ORF, was treated with DpnIand gel-purified to eliminate the template plasmid. The PCR product wasthen transformed into DH10ß E. coli cells containing bacmid bMON14272(Invitrogen) and Lambda RED recombinase-producing plasmid pKD46(Datsenko & Wanner, 2000) prepared in the above mentioned manner. In thefinal step, colonies resistant to kanamycin were subjected to PCR-basedanalysis using primers 75834 and 76420 (Table I) to verifyinsertion/elimination of the cat gene from the bacmid backbone. Positiveclones were further verified by DNA-sequencing of the obtained PCRproducts. According to this protocol, an internal part (498 nt=166 aa)of the vp39 ORF was removed, coordinates: 75894-76391 as indicated inFIG. 9.

Construction and Analysis of Repaired Vp39-Null Bacmids

To prepare a vp39 repair donor vector, we modified plasmid pFB-egfp(noted above) by introduction of the vp39 ORF under control of thepolyhedrin promoter. Initially, a 1073-bp fragment was amplified usingprimers vp39-SacI-F and vp39-XbaI-R (see Table I for primer sequences)from the bMON14272 template, and cloned into vector pJet1.2/Blunt(Fermentas) to form pJet1.2-vp39. After DNA sequence verification, thevp39 ORF was excised from pJet1.2-vp39 by SacI/XbaI double digestion,and then subcloned into SacI/XbaI-digested and gel-purified pFB-egfp togenerate donor plasmid pFB-egfp-vp39. After an unsuccessful attempt torescue AcMNPV vp39null with pFB-egfp-vp39, a set of novel donor plasmidswas prepared. First, a 2498-bp fragment containing vp39 and lef-4 ORFswas PCR-generated using primers vp39-StuI-F and lef-4-XbaI-R from bacmidbMON14272 template, and cloned into vector pJet1.2/Blunt (Fermentas) toform pJet1.2-vp39-lef-4. After DNA sequence confirmation, the fragmentcontaining vp39 and lef-4 ORFs was excised from pJet1.2-vp39-lef-4 byStuI/XbaI double digestion, and then subcloned into StuI/XbaI-digestedand gel-purified pFB-egfp to generate donor plasmid pFB-egfp-vp39-lef-4.

Parallelly, donor plasmid pFB-egfp-vp39-cg30 was constructed, where bothvp39 and cg-30 ORFs are driven from the very late polyhedrin promoter,and the cg-30 ORF can also use its native promoter situated inside the3′-end of the vp39 ORF. Briefly, a 1868-bp fragment carrying both vp39and cg-30 ORFs was amplified using primers cg30-XbaI-F and vp39-XbaI-R(noted above) and cloned into pJet1.2/Blunt, to generatepJet1.2-vp39-cg30. The vp39/cg-30 cassette was subcloned as SacI/Xbainto pFB-egfp, to create pFB-egfp-vp39-cg30. Additionally, a similardonor vector pFB-egfp-FLAG-vp39-cg30 was constructed, where vp39 ORF isN-terminally FLAG-tagged. The same strategy was employed to develop thisvector, only the reverse primer vp39-FLAG-SacI-R was used to amplifyvp39/cg-30 cassette instead of the vp39-XbaI-R primer.

All developed donor plasmids were transposed into vp39-null bacmidfollowing the Bac-to-Bac kit protocol (Invitrogen) and screened asdetailed above for vp80 repair bacmids. The functional analysis wasperformed as described above for the vp80 constructs.

Generation and Analysis of AcMNPV Vp1054-Null Bacmid

To verify the essential role of the vp1054 gene in the context of viralprogeny production and nucleocapsid assembly, we constructed an AcMNPVbacmid (bMON14272) with a deletion of vp1054 by homologous recombinationin E. coli according to the same procedure as for the vp80null bacmidconstruct with minor alternations. Since the vp1054 ORF is overlappingwith the essential lef-10 ORF, we could not remove the whole vp1054 ORF,but only a 955-bp nucleotide 3′-end part of the ORF. To preventtranslation of the C-truncated VP1054 mutant in insect cells, we decidedto mutate the first translation codon ATG→Met to ACG→Thr. This singlenucleotide substitution also changed an internal codon no. 32 (AAT) toAAC of lef-10 ORF, however, both are encoding the same amino acid (Asn).To accomplish this, we first amplified the 5′-end of the vp1054 ORFusing primers vp1054-KO-F and vp1054-KO-R1 from bacmid bMON14272(Invitrogen). The 214-bp PCR product contained a mutation of the ATGstart codon of the vp1054 ORF, introduced a synthetic stop/poly-A signalsequence for the lef-10 ORF, and has a 3′-end sequence homology overhangto the cat cassette to facilitate the second PCR, and a 49-bp homologysequence to the 5′-end of vp1054 ORF to mediate Lambda RED-directedhomologous recombination in E. coli. After gel-purification and DNAquantification, the 214-bp fragment was used as forward primer in asecond step PCR with reverse primer vp1054-KO-R2 with a plasmidcomprising a cat gene flanked with mutant LoxP sites as template. Theresulting 1230-bp PCR fragment, which contained the cat gene flanked bymutant LoxP sites, a mutated 5′-end of the vp1054 ORF and ˜50-bpsequences homologous to the 5′ or 3′ proximal region of the vp1054 ORF,was treated with DpnI and gel-purified to eliminate the templateplasmid. Recombination of this PCR product with the bMON14272 bacmid wasperformed as described above for the vp80 mutant. Kanamycin resistantcolonies were verified by PCR with primer pairs cat-F/cat-R,45510/46235, and 45122 and 46441 to check the insertion/elimination ofthe cat gene from the bacmid backbone. Insertion sites were alsoconfirmed by DNA-sequencing. This method resulted in the deletion of 955bp from nucleotide positions 45365 to 46319 in the AcMNPV clone C6genome provided in SEQ ID NO: 1. All primer sequences are given in Table1.

Construction of a Repaired Vp1054-Null Bacmid Construct

To prepare vp1054 repair donor vector, we modified plasmid pFB-egfp(noted above) by removing the polyhedrin promoter and replacing it witha fragment containing the vp1054 promoter region and the vp1054 ORF.First, a 1714-bp fragment containing both the vp1054 promoter and ORFsequence was amplified using primers vp1054-Rep-F and vp1054-Rep-R frombacmid bMON14272 template, and cloned into vector pJet1.2/Blunt(Fermentas) to form pJet1.2-pvp1054-vp1054. After DNA sequenceverification, the vp1054 cassette was excised frompJet1.2-pvp1054-vp1054 by StuI/XbaI double digestion, and then subclonedinto Bst1107I/XbaI-digested and gel-purified pFB-egfp to generate donorplasmid pFB-egfp-pvp1054-vp1054. The developed donor plasmids weretransposed into the vp1054-null bacmid following the Bac-to-Bac protocol(Invitrogen) and screened. Recombinant bacmids were analyzed as detailedabove for vp80 bacmids.

Generation and Analysis of AcMNPV p6.9-Null Bacmid

To verify the essential role of p6.9 in the context of viral progenyproduction, we constructed an AcMNPV bacmid (bMON14272) with a deletionof p6.9 by homologous recombination in E. coli. To accomplish this, achloramphenicol resistance gene (cat) flanked by mutant LoxP sites wasamplified using PCR primers p6.9-KO-F and p6.9-KO-R from a plasmidcomprising a cat gene flanked by mutant LoxP sites. Mutant viruses wereobtained following the same procedure as for the other mutants. For thePCR-based analysis of the finally obtained mutant clones the primerpairs cat-F and cat-R and 86596 and 86995 were used to checkinsertion/elimination of cat gene from bacmid backbone. Positive cloneswere also confirmed by DNA-sequencing. This method results in thedeletion of 164 bp from nucleotide positions 86716 to 86879 in theAcMNPV clone C6 genome provided in SEQ ID NO: 1. Table 1 for primersequences.

Construction and Functional Analysis of Repaired p6.9-Null Bacmids

To prepare p6.9 repair donor vectors, the pFB-GFP-p6.9 vector was used,which was constructed by Marcel Westenberg (Wageningen University). Tomake this vector, the AcMNPV p6.9 promoter sequence was amplified fromthe plasmid pAcMP1 (Hill-Perkins & Possee, 1990) with primers pp6.9-Fand pp6.9-R using the high-fidelity Expand long-template PCR system(Roche). The PCR product was cloned as SalI fragment into pFastBac1(Invitrogen), from which the polyhedrin promoter was deleted in advanceby fusing the Bst1107I to the StuI site, to obtain pFB1-p6.9. The p6.9promoter from pFB1-p6.9 was recloned as SnaBI/BamHI fragment into theBst1107I and BamHI sites of pFastBacDUAL (Invitrogen), thereby deletingthe polyhedrin promoter. Subsequently, the egfp reporter gene was cloneddownstream of the p10 promoter into the XmaI site to obtainpFB-GFP-p6.9. Finally, the p6.9 genes of AcMNPV and Spodoptera exigua(Se)MNPV were PCR amplified from either the AcMNPV bacmid (bMON14272) orSeMNPV genomic DNA by using the high-fidelity Expand long-template PCRsystem and primers generating EcoRI and NotI at the 5′ and 3′ ends,respectively (Table 1). The PCR products were cloned downstream of thep6.9 promoter in the EcoRI/NotI sites of pFB-GFP-p6.9. All generatedclones were sequenced to verify the incorporated p6.9 sequences.

The expression cassettes of both developed donor plasmids weretransposed into the p6.9-null bacmid following the Bac-to-Bac protocol(Invitrogen). Screening of transposition-positive constructs into thepolh locus was done by the triplex PCR-based assay as described abovefor the vp80 constructs. The analysis was performed as for the vp80constructs

Results

Silencing of AcMNPV Vp80 does not Affect Baculovirus Very Late GeneExpression

We explored the effect of transfecting Sf9 cells with different dsRNAsduring infection with AcMNPV-GFP. To trigger dsRNA-induced silencing ofselected baculoviral genes (vp1054, vp39, vp80, dbp and odv-ec27), wegenerated gene-specific dsRNAs using in vitro T7 RNA polymerase-basedsynthesis. However, when we began these studies it was not clear whatamount and time point of dsRNA transfection is the most effective tosilence baculoviral genes. To determine an optimal amount of dsRNA forRNAi assay purposes in baculovirus-infected cells, we first attempted tosilence reporter egfp gene with different amounts of dsRNA. These pilotassays showed that the most potent RNAi effect is achieved using 100 pgdsRNA per cell (data not shown). At the same time, it was also provedthat RNAi treatment has no negative effect on the production ofinfectious budded virions progeny. We also tried to transfect dsRNA intothe cells at two different time points, 24 h prior to infection or 1 hp.i. The results proved that transfection performed at 1 h p.i. is moreefficient in silencing of genes expressed at late/very late phases ofbaculoviral infection in contrast to transfection carried out at 24 hprior to infection (data not shown). In addition, to ensure thatknock-down was gene-specific, dsRNA corresponding to the cat gene wastransfected as an RNAi negative control. Herein, we could observe amoderate inhibition of baculovirus infection propagation in comparisonto untransfected insect cells. However, the same phenomenon was alsoobserved when insect cells were treated only with transfection reagents.Therefore, we could conclude that the effect can be explained by anegative impact (cytotoxicity) of the presence of transfection reagentson cell viability.

Silencing screening of baculovirus genes revealed that down-regulationof vp1054, vp39, dbp and odv/ec-27 is also associated with a reductionor inhibition of very late gene expression measured by EGFP detection(FIGS. 1A and 1B). The highest levels of this inhibition were observedin dbp- and odv/ec-27-targeted cells. The cause of this effect can beexplained by the presence of bi-cistronic and overlapping mRNAtranscripts, which are produced during a baculovirus replication cycle.Eventually, a cross-reaction with targets of limited sequencesimilarities can also be involved in the process. Only cells treatedwith vp80 dsRNA showed a similar level of EGFP expression asuntransfected cells or particularly with cat dsRNA-treated cells.Importantly, very few EGFP-producing cells were observed in insect cellswhere egfp-specific dsRNA was introduced (positive RNAi control),showing that the transfection efficiency was high. Based on our RNAiscreening achievements, the vp80 gene (locus) seems to be a suitablecandidate for RNAi-based targeting in context of interference withbaculoviral very late gene expression.

Knock-Down of Vp80 Totally Prevents Production of BVs and NormallyAppearing ODVs

To determine the roles of selected candidate genes (vp1054, vp39, vp80,dbp and odv/ec-27) in production of budded virions progeny, cell culturemedium (36 h p.i.) from dsRNA-treated cells was examined for thepresence of BVs. End-point dilution-based titrations confirmed that alltested genes are essential for infectious budded virus progenyproduction (FIG. 1C). We were not able to detect any infectious BVs invp80- and dbp-targeted cells. In addition, PCR-based assay indicatedthat defective or non-infectious viral particles are also not producedin vp80-targeted cells. It is important to point out that the resultsalso showed a significant decrease in the production of infectious BVsin the RNAi controls (egfp- and cat-specific dsRNA-treated cells)compared to untransfected cells. The cytotoxicity of transfectionreagents is again the assumed cause of this negative effect. Electronmicroscopy analysis of cell lysates showed that formation of ODVs androd-like structures was totally inhibited in cells treated withdsRNA-vp39 as expected (FIG. 1D). Production of ODVs and rod-likestructures was also significantly reduced in insect cells treated withdsRNA-vp80 (FIG. 1D). However, in vp80-targeted cells we could mostlyfind nucleocapsids of aberrant phenotypes (pointed shape). On the otherhand, introduction of dsRNA-cat into insect cells did not cause anychanges in the production of ODVs.

The AcMNPV Vp80 Gene is Essential for Viral Replication

An AcMNPV deletion virus was constructed as detailed in FIG. 2. Repairconstructs were designed such that the wild-type vp80 ORF or N- andC-terminally FLAG-tagged vp80 genes along with its native or polyhedrinpromoter regions were inserted into the polyhedrin locus along with theegfp gene under the p10 promoter (FIG. 3A). To investigate the functionof the vp80 gene, Sf9 cells were transfected with either the knock-outor repair bacmid constructs and monitored for EGFP expression byfluorescence microscopy. When Ac-vp80 null was introduced into Sf9cells, no viral propagation was observed in cell culture at 72 h to 120h p.t. We could observe only a “single-cell infection” phenotype similarto the phenotype of Ac-gp64null bacmid (FIG. 3B). The results indicatethat Ac-vp80null is able to reach the very late phase of infection asconfirmed by p10 promoter-driven EGFP expression. From 72 h to 120 hoursp.t., widespread EGFP expression could be seen in insect cell monolayersthat were transfected with the three repair (vp80 driven from its nativepromoter, vp80 driven from polyhedrin promoter and N-terminallyFLAG-tagged vp80 driven from its native promoter) constructs indicatingthat these bacmids were able to produce levels of infectious buddedvirions sufficient to initiate secondary infection at a similar level asthe wild-type bacmid (FIG. 3B). In contrast, in insect cells transfectedwith C-terminally FLAG-tagged vp80 repair constructs, by 72 h p.t. EGFPexpression was only observed in isolated cells that were initiallytransfected indicating that this bacmid construct is defective in viralreplication (FIG. 3B). However, by 96 h p.t. formation of tiny plaqueswas observed and by 120 h p.t. very few plaques of normal size weredeveloped. The results show that the C-terminally flagged mutant isstrongly delayed in producing budded virus and showed that an unmodifiedC-terminus is very important for the function of VP80. At 5 days p.t.,cell culture supernatants were removed and added to freshly plated Sf9cells and then incubated for 3 days to detect infection by virusgenerated from cells transfected with these bacmids. As expected, Sf9cells incubated with supernatants from the transfections with repairconstructs showed numerous EGFP expressing cells (FIG. 3C).Nevertheless, cells incubated with supernatant from C-terminallyFLAG-tagged constructs showed a significant reduction in the number ofEGFP-positive cells. On the other hand, in insect cells incubated withsupernatant from the transfection with the vp80 knockout, no EGFPexpression was detected at any time-point analyzed up to 72 h (FIG. 3C).

Moreover, to characterize the exact effect of deletion of the vp80 geneon AcMNPV infection, the viral propagation in transfected Sf9 cells wascompared between Ac-wt, Ac-Δvp80, Ac-Δvp80-vp80Rep,Ac-Δvp80-polh-vp80Rep, Ac-Δvp80-FLAG-vp80Rep and Ac-Δvp80-vp80-FLAGRep.Cell culture supernatants of all the above bacmid constructs wereanalysed at indicated time points for BV production (FIG. 4). Asexpected, the repaired Ac-Δvp80-vp80Rep, Ac-Δvp80-polh-vp80Rep,Ac-Δvp80-FLAG-vp80Rep viruses showed kinetics of viral replicationconsistent with wild-type virus (Ac-wt) propagation. Budded virionproduction by the C-terminally flagged Ac-Δvp80-vp80-FLAGRep virus wasreduced to approximately 0.06% compared to the Ac-wt virus or the otherrepaired viruses.

These results indicate that the vp80 gene is essential for infectious BVproduction. It has clearly been proven that the whole sequence of vp80ORF can completely be deleted from the bacmid backbone and adequatelyrescued by introduction of the vp80 ORF into a heterologous site(polyhedrin locus) of the genome. We also showed that vp80 geneexpression can be driven by the heterologous polyhedrin promotersequence with no negative effect on viral replication in cell culture.Additionally, we observed that the N-terminus in contrast to theC-terminus of VP80 is permissive to gene modifications (epitopetag-labeling). We noted that the kinetics of the C-terminallyFLAG-tagged VP80 virus was significantly delayed when compared with allother rescue or wild-type viruses, indicating the functional importanceof the VP80 C-terminus.

VP80 is Required for Production of Both BV and ODV

The results described above indicated that the Ac-vp80null mutant iscompletely defective in production of infectious budded virus. However,there was also a possibility that the mutant can still producenon-infectious budded particles. To investigate the ability, Sf9 cellswere transfected with either the knock-out, repair or wild-type bacmidconstructs and 7 days p.t. cell culture mediums were ultracentrifuged topellet budded viruses. The formed pellets were either analyzed bynegative staining electron microscopy or by Western blot- and PCR-baseddetection to confirm the presence of the budded viruses. No intactbudded virus, virus-like particles, nor its structures (such as majorcapsid protein VP39 and viral genome sequence) were revealed in thepellet from the cells transfected with the Ac-vp80null mutant (FIGS. 5Aand 5B). On the other hand, all analyzed repair constructs producednormally-appearing budded virus as compared with budded virus-derivedfrom the wild-type virus (FIG. 5A). Nevertheless, it was very difficultto find representative budded virions in the pellet derived fromC-terminally FLAG-tagged vp80 gene repair construct-transfected cells.

To further characterize deletion of the vp80 gene on baculovirus lifecycle, electron microscopy was performed with ultra-thin sectionsgenerated from bacmid-transfected cells. The Ac-vp80null-transfectedcells developed the typical phenotype of baculovirus-infected cells withan enlarged nucleus, a fragmented host chromatin, an electron-densevirogenic stroma, etc. (FIG. 6A). The absence of VP80 did not preventformation of normally-appearing nucleocapsids inside the virogenicstroma (FIG. 6C). The formed nucleocapsids were phenotypicallyundistinguishable from those produced by either the Ac-vp80null repairor Ac-wt bacmids. However, an abundance of assembled nucleocapsids wasrather less as compared with cells transfected with the Ac-vp80nullrepair or Ac-wt bacmids (FIGS. 6E and 6G). In addition, noocclusion-derived virions nor bundles of nucleocapsids prior to anenvelopment could be observed in the peristromal compartment of anucleoplasm (so called the ring zone) of Ac-vp80null bacmid-transfectedcells (FIGS. 6B and 6D). It seems that VP80 plays a role duringmaturation of nucleocapsids and/or their release/transport from thevirogenic stroma. Eventually, VP80 can somehow contribute to anefficient nucleocapsid assembly which could be explained by the smallnumber of nucleocapsids present in the virogenic stroma of Ac-vp80nulltransfected cells. When the vp80 gene was re-introduced back into thebacmid mutant, a lot of nucleocapsids and occlusion-derived virionscould be seen in the ring zones of transfected cells (FIG. 6F). Anabundance and morphology of occlusion-derived virions produced inAc-Δvp80-vp80 repair bacmid-transfected cells were similar to thoseproduced by wild-type bacmid (FIGS. 6F and 6H).

VP80 Function can be Complemented by the Trans-Acting Vp80 Gene

To prove that VP80 function can be complemented by the trans-acting vp80ORF, a complementation assay was performed with a transgenic cell line,Sf9-vp80, that was stably transformed with the vp80 gene expressed undercontrol of an early baculovirus Orgyia pseudotsugata ie-2 promoter. Inthe assay, both Sf9 and Sf9-vp80 cells were transfected with theAc-vp80null bacmid mutant (FIG. 7). Virus infection spread was monitoredby EGFP-specific fluorescence at 72 h and 96 h p.t. In Sf9-vp80 cells wecould observe viral plaques demonstrating the virus spread. On the otherhand, in Sf9 cells only “single-cell infection” phenotype could be seenas previously described above. After six days, the cell culturesupernatants were harvested and used as an inoculum to infect freshgroups of Sf9 cells. After 5 days, EGFP-positive cells were monitored byfluorescence microscopy. Only “single-cell infection” phenotype wasobserved in Sf9 cells receiving the supernatant from Sf9-vp80 cells. Asassumed, no EGFP signal was detected in Sf9 cells receiving thesupernatant from Sf9 cells. These results show that the Ac-vp80null canbe rescued by VP80-expressing cells (Sf9-vp80) and demonstrate that theobserved complementation is due to VP80 protein expressed from the hostcell line and not from acquisition of the vp80 gene from the cell line.In other words, the results match requirements asked to producebiopharmaceuticals (EGFP protein in our model assay) withoutcontaminating baculovirus virions.

Generation and Characterization of Vp39-Null Bacmid

To study the functionality of the AcMNPV vp39 gene during virusinfection, a vp39-null AcMNPV bacmid was constructed by partial deletionof the vp39 gene. The deletion construct was selected by its resistanceto chloramphenicol indicating that site-specific deletion of the vp39gene had occurred. In the resulting vp39-null AcMNPV bacmid, theinternal part of vp39 gene was correctly replaced by the cat gene.Subsequently, the cat was eliminated by Cre/LoxP recombination (FIG.8A). The vp39 sequence was removed from nucleotides 75894 to 76391according to the AcMNPV clone C6 genome sequence (SEQ ID NO:1). Thestructure of the vp39 deletion constructs was confirmed by PCR usingprimers 75834 and 76420 (FIG. 8B). A 647-bp DNA fragment was amplifiedwhen wild-type AcMNPV bacmid was used as a template, whereas a 1113-bpDNA fragment could be amplified on AcMNPV vp39-null(+cat) template (FIG.8B). When the final construct AcMNPV-vp80null (−cat) with eliminated catcassette was used in PCR analysis, only a short 183-bp DNA fragmentcould be detected (FIG. 8B). The results were confirmed by DNAsequencing.

Functional mapping of vp39 ORF indicates a presumable functionalrelationship between vp39 and cg-30 ORFs

The repair constructs were designed in such a way that the wild-typevp39 ORF under control of the polyhedrin promoter sequence was insertedinto the polyhedrin locus along with the egfp gene controlled by the p10promoter (FIG. 9A). To investigate the function of the vp39 gene, Sf9cells were transfected with either the knock-out or repair bacmidconstructs and monitored for EGFP expression by fluorescence microscopy.When Ac-vp39 null was introduced into Sf9 cells, no viral propagationwas observed from 72 h to 168 h p.t. We could observe only a“single-cell infection” phenotype similar to the phenotype of theAc-gp64null bacmid (FIG. 9B).

These results indicate that the Ac-vp39null construct is able to reachthe very late phase of infection as shown by the p10 promoter-drivenEGFP expression. Unexpectedly, no viral propagation could be seen ininsect cell monolayers that were transfected with the vp39 repair (vp39driven from polyhedrin, Ac-Δvp39-polh-vp39Rep) constructs (FIG. 3B). Forthis reason, we decided to prepare three extra repair bacmids carryingboth vp39 and lef-4 ORFs under control of their native promoters. Whenthe insect cells were transfected with these repair constructs againviral replication did not occur (FIG. 9B) and a “single-cell infection”phenotype was observed from 72 h to 168 h p.t. Interestingly, in insectcell monolayers that were transfected with the repair constructscarrying both vp39 (or FLAG-tagged vp39) and cg-30 we could observe tinyclusters of EGFP-positive cells (3-5 cells) (FIG. 9B). However, we didnot see a full-value viral replication as that of the wild-type vector(Ac-wt),

At 7 days p.t., cell culture supernatants were collected and added tofreshly plated Sf9 cells, which were then incubated for 3 days to detectinfection by virus generated from cells transfected with all bacmidsmentioned here (FIG. 9C). As expected, Sf9 cells incubated with thesupernatant from Ac-wt transfections showed numerous EGFP expressingcells. On the other hand, cells incubated with supernatants fromAc-Δvp39-polh-vp39Rep and Ac-Δvp39-vp39-lef-4Rep constructs did not showany EGFP-positive cells. However, in insect cells incubated withsupernatants from Ac-Δvp39-vp39-cg30Rep and Ac-Δvp39-FLAG-vp39-Rep, anumber of EGFP-expressing cells were detected (FIG. 9C). These resultsindicated that a possible functional relationship between the vp39 andcg-30 ORFs is required for baculovirus replication.

Since the vp39 ORF sequence overlaps with the promoter sequences of thetwo flanking ORFs (lef-4 and cg-30), we could not delete the whole vp39ORF in our vp39null bacmid construct. It may therefore also be that C-and/or N-truncated mutant(s) of vp39 may be expressed which mayinterfere as a competitive inhibitor with the normal VP39 protein.

Construction and Analysis of Vp1054-Null Bacmid

To study the functionality of the AcMNPV vp1054 gene during virusinfection, a vp1054-null AcMNPV bacmid was constructed by partiallydeleting the vp1054 gene from AcMNPV bacmid (bMON14272) by homologousrecombination in E. coli. The deletion construct was selected by itsresistance to chloramphenicol that indicated that site-specific deletionof the vp1054 gene had occurred. In the resulting vp1054-null AcMNPVbacmid, the 955-bp 3′-end part of the vp1054 gene was correctly replacedby the cat gene. Subsequently, the antibiotic resistance cassette (cat)was eliminated from bacmid backbone using Cre/LoxP recombination system(FIG. 10A). The deleted sequence was removed from the nucleotidecoordinates 45365 to 46319 according to the AcMNPV clone C6 genomesequence (SEQ ID NO:1). The structure of all the deletion constructs wasconfirmed by PCR (FIG. 10B). When the vp1054 gene is present, as in theparental wild-type AcMNPV bacmid, a 775-bp PCR product can be amplifiedusing primers 45510 and 46235, whereas a 596-bp PCR fragment amplifiedwith cat-F and cat-R primers is produced only when the cat gene wasintroduced into the bacmid sequence in case of AcMNPV vp1054null (+cat)construct (FIG. 10B). Correct recombination process was also confirmedby PCR mapping of vp1054 locus using primers 45122 and 46441. A 1320-bpDNA fragment was amplified when wild-type AcMNPV bacmid was used as atemplate, whereas a 1353-bp DNA fragment could be amplified on AcMNPVvp1054-null(+cat) template (FIG. 10B). When final constructAcMNPV-vp1054null (−cat) with eliminated cat cassette was used in PCRanalysis, only a 423-bp DNA fragment could be detected (FIG. 10B).Positive clones were successfully verified by DNA sequencing.

AcMNPV Vp1054 Gene is Essential for Viral Replication

The repair construct was designed such that the AcMNPV vp1054 ORF withits native promoter region was inserted into the polyhedrin locus alongwith the egfp gene under the control of the p10 promoter (FIG. 11A).Since the vp1054 promoter and ORF sequences are overlapping with lef-10ORF, the repair construct is also capable to express LEF-10. Toinvestigate the function of the vp1054 gene, Sf9 cells were transfectedwith either the vp1054 knock-out or repair bacmid construct andmonitored for EGFP expression by fluorescence microscopy. When Ac-vp1054null construct was introduced into Sf9 cells, no viral propagation wasobserved in cell culture at 72 h to 120 h p.t. We could observe only a“single-cell infection” phenotype similar to the phenotype ofAc-gp64null bacmid (FIG. 11B). The results indicate that Ac-vp1054nullis able to reach the very late phase of infection as confirmed by p10promoter-driven EGFP expression. In other words, the results suggestthat the expression of late expression factor 10, LEF-10, was notaffected in vp1054-null bacmid mutant. From 72 h to 120 hours p.t.,widespread EGFP expression could be seen in insect cell monolayers thatwere transfected with the repair constructs (Ac-Δvp1054-vp1054). Theresults are indicating that the repair bacmid is able to produce levelsof infectious budded virions sufficient to initiate secondary infectionat a similar level as the wild-type bacmid (FIG. 11B). At 6 days p.t.,cell culture supernatants were removed and added to freshly plated Sf9cells and then incubated for 3 days to detect infection by virusgenerated from cells transfected with these bacmids. As expected, Sf9cells incubated with supernatants from the transfections with the repairconstructs showed numerous EGFP expressing cells (FIG. 11C). On theother hand, in insect cells incubated with supernatant from thetransfection with the Ac-vp1054null knockout, no EGFP expression wasdetected at any time-point analyzed up to 72 h (FIG. 11C).

These results indicate that the vp1054 gene is essential for infectiousBV production. It has clearly been proven that the 955-bp 3′-endsequence part of the vp1054 ORF can completely be deleted from thebacmid backbone and adequately rescued by introduction of the AcMNPVvp1054 ORF into a heterologous site (polyhedrin locus) of the genome. Inaddition, the results proved that deletion of the vp1054 gene does notaffect very late gene expression, as demonstrated by EGFP-positive cellsin cells transfected with Ac-vp1054null bacmid mutant (FIG. 11B).

Generation and Characterization of p6.9-Null Bacmid

To study the functionality of the AcMNPV p6.9 gene during virusinfection, a vp80-null AcMNPV bacmid was constructed by deleting thep6.9 gene from AcMNPV bacmid (bMON14272) by homologous recombination inE. coli. The deletion construct was selected by its resistance tochloramphenicol that indicated that site-specific deletion of the p6.9gene had occurred. In the resulting p6.9-null AcMNPV bacmid, the p6.9gene was correctly replaced by the cat gene. Subsequently, theantibiotic resistance cassette (cat) was eliminated from bacmid backboneusing Cre/LoxP recombination system (FIG. 12A). The deleted sequence wasremoved from the translational start codon (ATG→Met) to the stop codon(TAT→Tyr), nucleotide coordinates 86716 to 86879 according to the AcMNPVclone C6 genome sequence (SEQ ID NO:1). The stop codon of the p6.9 orfwas not removed since its sequence is overlapping with the stop codon offlanked lef-5 orf. The structure of all the deletion constructs wasconfirmed by PCR (FIG. 12 B). When the p6.9 gene is present, as in theparental wild-type AcMNPV bacmid, a 596-bp PCR fragment could be onlyamplified with cat-F and cat-R primers when cat gene was introduced intobacmid sequence in case of AcMNPV p6.9null (+cat) construct (FIG. 12B).Correct recombination process was also confirmed by PCR mapping of p6.9locus using primers 86596 and 86995. A 400-bp DNA fragment was amplifiedwhen wild-type AcMNPV bacmid was used as a template, whereas a 1220-bpDNA fragment could be amplified on AcMNPV vp80-null(+cat) template (FIG.12B). When final construct AcMNPV-vp80null (−cat) with eliminated catcassette was used in PCR analysis, only a short 290-bp DNA fragmentcould be detected (FIG. 12B). Positive clones were successfully verifiedby DNA sequencing.

AcMNPV p6.9 Gene is Essential for Viral Replication

The repair constructs were designed such that the wild-type AcMNPV orSeMNPV p6.9 ORFs with AcMNPV p6.9 promoter region were inserted into thepolyhedrin locus along with the egfp gene under the p10 promoter (FIG.13A). To investigate the function of the p6.9 gene, Sf9 cells weretransfected with either the p6.9 knock-out or repair bacmid constructsand monitored for EGFP expression by fluorescence microscopy. WhenAc-p6.9 null was introduced into Sf9 cells, no viral propagation wasobserved in cell culture at 72 h to 120 h p.t. We could observe only a“single-cell infection” phenotype similar to the phenotype ofAc-gp64null bacmid (FIG. 13B). The results indicate that Ac-p6.9null isable to reach the very late phase of infection as confirmed by p10promoter-driven EGFP expression. From 72 h to 120 hours p.t., widespreadEGFP expression could be seen in insect cell monolayers that weretransfected with the two repair constructs (Ac-Δp6.9-Acp6.9 andAc-Δp6.9-Sep6.9). The results are indicating that these two repairbacmids are able to produce levels of infectious budded virionssufficient to initiate secondary infection at a similar level as thewild-type bacmid (FIG. 13B). At 6 days p.t., cell culture supernatantswere removed and added to freshly plated Sf9 cells and then incubatedfor 3 days to detect infection by virus generated from cells transfectedwith these bacmids. As expected, Sf9 cells incubated with supernatantsfrom the transfections with the repair constructs showed numerous EGFPexpressing cells (FIG. 13C). On the other hand, in insect cellsincubated with supernatant from the transfection with the Ac-p6.9nullknockout, no EGFP expression was detected at any time-point analyzed upto 72 h (FIG. 3C). Moreover, to characterize the exact effect ofdeletion of the p6.9 gene on AcMNPV infection, the viral propagation intransfected Sf9 cells was compared between Ac-wt, Ac-Δp6.9,Ac-Δp6.9-Acp6.9Rep, and Ac-Δp6.9-Sep6.9Rep). Cell culture supernatantsof all the above bacmid constructs were analysed at indicated timepoints for BV production (FIG. 13D). As expected, the repairedAc-Δp6.9-Acp6.9Rep and Ac-Δp6.9-Sep6.9Rep viruses showed kinetics ofviral replication consistent with wild-type virus (Ac-wt) propagation.

These results indicate that the p6.9 gene is essential for infectious BVproduction. It has clearly been proven that the whole sequence of p6.9ORF can completely be deleted from the bacmid backbone and adequatelyrescued by introduction of the AcMNPV vp80 ORF into a heterologous site(polyhedrin locus) of the genome. We also showed that p6.9 gene can becomplemented efficiently by the SeMNPV-derived p6.9 ORF (M. Westenberg).In addition, the results proved that deletion of the p6.9 gene does notaffect very late gene expression, as demonstrated by EGFP-positive cellsin cells transfected with Ac-p6.9null bacmid mutant (FIG. 15B).

Example II

The inventors have amended the best mode of the present invention in thefollowing example.

Materials and Methods Generation of an Antibiotic Resistance Gene-FreeAcMNPV Vp80-Null Bacmid

To determine whether the VP80 protein has an essential role in thecontext of viral progeny production, we constructed an AcMNPV bacmid(derived from bMON14272 (from Invitrogen)) with a deletion of the vp80ORF by homologous recombination in E. coli. To accomplish this, a catgene flanked by mutant LoxP sites (Suzuki et al., 2005) was amplifiedusing PCR primers vp80-KO-F and vp80-KO-R (see Table 1) from a plasmidcomprising a cat gene flanked by mutant LoxP sites. The resulting PCRfragment, which contained the cat gene flanked by mutant LoxP sites andAcMNPV ˜50-bp homology sequences to the 5′ or 3′ proximal region of thevp80 ORF, was treated with DpnI and gel-purified to eliminate thetemplate plasmid. The PCR product was then transformed into DH101 E.coli cells containing bMON14272 (Invitrogen) and the Lambda REDrecombinase-producing plasmid pKD46 (Datsenko & Wanner, 2000), which hadbeen prepared in the following manner. Transformed DH10ß-bMON14272/pKD46E. coli cells were grown in 50-ml LB (2.0% peptone, 0.5% yeast extract,85.5 mM NaCl, [pH 7.0]) cultures with kanamycin (50 μg/ml), ampicillin(100 μg/ml) and L-arabinose (1.5 mg/ml) at 30° C. to an OD₆₀₀ of ≈0.6and then made electrocompetent by a standard procedure. Theelectroporated cells were incubated at 37° C. for 3 h in 3 ml LB mediumand plated on LB-agar containing chloramphenicol at a concentration of6.5 μg/ml. After 48-h incubation at 37° C., thechloramphenicol-resistant colonies were streaked to fresh LB-agar mediumwith 34 μg/ml chloramphenicol. The plates were incubated at 37° C.overnight, and colonies resistant to chloramphenicol were selected forfurther confirmation of the relevant genotype by PCR. Primers 90292 and90889 were used to confirm the absence of the vp80 ORF, and primerscat-F and cat-R were employed to verify the presence of cat cassetteinto bacmid (detailed sequences in Table 1).

To eliminate the introduced antibiotic resistance gene (cat) from thebacmid backbone, a Cre/LoxP recombinase system was employed. A Crerecombinase-carrying plasmid pCRE obtained from Jeanine Louwerse (LUMCLeiden, The Netherlands) was introduced into DH10b-bMON14272-vp80null E.coli cells, and CRE expression was subsequently induced by the additionof isopropyl thiogalactoside (IPTG). Briefly, the electroporated cellswere incubated at 37° C. for 3 h in 3 ml of LB medium (2.0% peptone,0.5% yeast extract, 85.5 mM NaCl, [pH 7.0]) and plated on LB-agar mediumcontaining 50 μg/ml kanamycin, 100 μg/ml ampicillin and 2 mM IPTG. After24-h incubation, colonies resistant to kanamycin and ampicillin wereselected for further verification of the desired genotype by PCR. InPCR-based analysis, primers 89507 and 91713 (Table 1) were used toverify elimination of cat gene from bacmid backbone. Positive cloneswere also confirmed by DNA-sequencing.

To recover transposition competence, the helper transposase-encodingplasmid pMON7124 (Invitrogen) was re-introduced intoDH10ß-bMON14272-vp80null E. coli cells. Finally, the egfp reporter genewas introduced into the vp80-null bacmid to facilitate observation ofits behaviour in insect cells. Briefly, the egfp reporter gene wasamplified using PCR oligonucleotides gfp-NheI-F and gfp-SphI-R (Table 1)from plasmid pEGFP-N3 (Clontech). The PCR product was cloned intoplasmid pJet1.2/Blunt using CloneJET™ PCR Cloning Kit (Fermentas)according to manufacturer's protocol. Subsequently, the egfp ORF wasexcised from error-free pJet1.2-egfp with NheI and SphI and subclonedinto NheI/SphI-digested pFastBacDUAL (Invitrogen), to generate plasmidpFB-egfp. An expression cassette containing the egfp reporter gene undertranscriptional control of the very late p10 promoter was transposedfrom pFB-egfp into polyhedrin locus of vp80-null bacmid as described inthe Bac-to-Bac manual (Invitrogen). In the resulting genome, thecomplete vp80 ORF has been removed (see FIG. 2). This corresponds to thedeletion of 2074 bp from nucleotide positions 89564 to 91637 in theAcMNPV clone C6 genome provided in SEQ ID NO: 1.

Construction of Repaired Vp80-Null Bacmids

To prepare vp80 repair donor vectors, we modified plasmid pFB-egfp(noted above) by removing the polyhedrin promoter and replacing it witha fragment containing the vp80 promoter region and the vp80 ORF. First,a 2300-bp fragment containing both the vp80 promoter and ORF sequencewas amplified using primers pvp80-StuI-F and vp80-XbaI-R (Table 1) frombacmid bMON14272 template, and cloned into vector pJet1.2/Blunt(Fermentas) to form pJet1.2-pvp80-vp80. After DNA sequence verification,the vp80 cassette was excised from pJet1.2-pvp80-vp80 by StuI/XbaIdouble digestion, and then subcloned into Bst1107I/XbaI-digested andgel-purified pFB-egfp to generate donor plasmid pFB-egfp-pvp80-vp80.Parallelly, a donor plasmid pFB-egfp-polh-vp80, where vp80 ORF is drivenby the very late polyhedrin promoter (polh) was constructed. To thisaim, a 2105-bp fragment carrying the vp80 ORF was amplified usingprimers vp80-SacI-F and vp80-XbaI-R (Table 1) and cloned intopJet1.2/Blunt, to generate pJet1.2-vp80. In the final step, the vp80 ORFwas cut out (SacI/XbaI) from pJet1.2-vp80, and subcloned intoSacI/XbaI-digested pFB-egfp, to create pFB-egfp-poIH-vp80.

To overcome a problem associated with the unavailability of anti-VP80antibody, FLAG tag decoration (N- and C-terminus fusion) of VP80 wasperformed to facilitate immunodetection. The N-terminally fusedFLAG-vp80 sequence was generated by a double-step PCR strategy, aso-called fusion PCR. First, a 259-bp fragment containing the vp80promoter and the FLAG tag was PCR amplified using primers pvp80-StuI-Fand vp80-FLAG-R1 from the bMON14272 bacmid template. Aftergel-purification and DNA quantification, the 259-bp fragment was used asforward primer in a second step PCR amplification with the reverseprimer vp80-XbaI-R on the bMON14272 bacmid template. The final PCRproduct (2324 bp) was cloned into vector pJet1.2/Blunt (Fermentas) toform pJet1.2-pvp80-FLAG-vp80. After DNA sequence verification, theFLAG-vp80 cassette was excised from pJet1.2-pvp80-FLAG-vp80 by StuI/XbaIdouble digestion, and then subcloned into Bst1107I/XbaI-digested andgel-purified pFB-egfp to generate donor plasmidpFB-egfp-pvp80-FLAG-vp80. The C-terminally fused vp80-FLAG cassette wasamplified using pvp80-StuI-F and vp80-FLAG-R from the bMON14272 bacmidtemplate. The 2324-bp fragment was cloned into pJet1.2/Blunt, andsubsequently transferred into pFB-egfp in a similar way as previousconstructs.

The inserts of all developed donor plasmids were transposed into thevp80-null bacmid following the Bac-to-Bac protocol (Invitrogen).Screening of transposition-positive constructs into the polh locus wasdone by a triplex PCR-based assay employing M13 forward and reverseprimers and a gentamicin resistance gene-specific primer GenR (Table 1).

Transfection-Infection Assay

Bacmid DNAs were prepared from 1.5-ml overnight bacterial cultures of 2to 3 independent colonies carrying the bacmid with the insertedheterologous gene according to the Bac-to-Bac manual (Invitrogen) andwere analyzed in parallel. For transfections, 1 μg of each bacmid DNApreparation was used to transfect 1×10⁶ Sf9 cells in a 6-well plate bythe Cellfectin™-based transfection protocol as described in theBac-to-Bac (Invitrogen) manual. From 72 h to 120 h post transfection(p.t.), viral propagation was checked by fluorescence microscopy. At 120h p.t., the cell culture medium was centrifuged for 5 min at 2000×g toremove cell debris, and this clarified supernatant was used to infect1.5×10⁶ Sf9 cells in 6-well plates. After 72 h p.i., the spread of virusinfection was again monitored by fluorescence microscopy. In allexperiments, a wild-type bMON14272 bacmid carrying the egfp reportergene under control of the p10 promoter was used as positive control. AbMON14272-gp64null bacmid also carrying the egfp reporter gene undercontrol p10 promoter served as negative control, since it has lost theability of cell-to-cell movement of the infection (Lung et al., 2002).

Time-Course Characterization of Viral Propagation in Cell Culture

Time course analyses were performed to compare budded virus productionof the AcMNPV-vp80null virus and the various repair constructs incomparison to the wild type AcMNPV bacmid (Ac-wt) all containing egfp.Briefly, the Sf9 cells were seeded in 6-well tissue culture plates(1×10⁶ cells/well in 1 ml Sf900-II culture medium without serum at 28°C.). After two hours, the culture medium was removed, and the cells weretransfected with 5 μg bacmid DNA, under standard conditions asrecommended in Bac-to-Bac manual (Invitrogen). Cell culture supernatantswere harvested at 24, 48, 72, 96 and 120 h p.t., and analysed for theproduction of infectious budded virus by an end-point dilution assay todetermine the tissue culture infective dose 50 (TCID₅₀). Infection wasdetermined by monitoring egfp expression (from the p10 promoter). Theaverage values of infectious titers derived from three independenttransfections were calculated and plotted into graphs.

Transmission Electron Microscopy

Insect Sf9 cells were seeded in a 25T flask (3.5×10⁶ cells/flask), andtransfected with 20 μg either the Ac-Δvp80, rescue Ac-Δvp80-vp80 orAc-wt bacmid construct. After 48 h p.t., the cells were harvested andprepared for transmission electron microscopy as described previously(van Lent et al., 1990). Samples were examined and photographed with aPhilips CM12 electron microscope.

Budded Virus Production Assay

Insect Sf9 cells were seeded in two 25T flasks (3.5×10⁶ cells/flask),and transfected with 20 μg either Ac-Δvp80, Ac-Δvp80-vp80,Ac-Δvp80-pH-vp80, Ac-Δvp80-FLAG-vp80, Ac-Δvp80-vp80-FLAG, or Ac-wtbacmid construct. Five days p.t., the BV-enriched cell culturesupernatants were harvested, and ultracentrifuged through a cushion of10% sucrose solution (25,000 rpm for 1.5 hour, Beckman SW32). Pelletedbudded virions were resuspended in sterile demi-water, and prepared foreither negative staining electron microscopy, SDS-polyacrylamideelectrophoresis, or PCR-based detection (as mentioned above).

Purification of ODVs and Rod-Shaped Structures from Infected Cells

The presence of ODVs and rod-like structures in infected/transfectedinsect cells was analyzed by electron microscopy (EM). For this purpose,insect cells were harvested 48 h p.i., lysed and the cell lysates wereultracentrifuged through a 40% sucrose cushion in TE (1 mM Tris-HCl pH7.4, 0.1 mM EDTA) buffer (45,000 rpm for 1 hour, Beckman SW55). Pelletswere resuspended in sterile demi-water and analyzed by negative stainingEM as described previously (van Lent et al., 1990).

Purification and Fractionation of BV and ODV Virions

To produce BVs, 3.0×10⁷ Sf9 cells were infected with Ac-Δvp80-Flag.vp80or control Ac-wt virus at an MOI=1. Six days p.i., 72 ml of BV-enrichedmedium was collected and centrifuged at 1,500×g for 10 min. Thesupernatant was then ultracentrifuged at 80,000×g (Beckman SW28 rotor)for 60 min at 4° C. The BV pellet was resuspended in 350 μl 0.1×TEbuffer, and loaded onto a linear sucrose gradient (25 to 56% (w/v)), andultracentrifuged at 80,000×g (Beckman SW55 rotor) for 90 min at 4° C.The formed BV band was collected and diluted in 12 ml 0.1×TE. The BVpreparation was concentrated at 80,000×g for 60 min at 4° C. The finalvirus pellet was resuspended in 150 μl of 0.1×TE.

To produce ODVs, 6.0×10⁷ Sf9 cells were co-infected withAc-Δvp80-Flag.vp80 (MOI=25) and AcMNPV (MOI=5) viruses (strain E2, Smith& Summers, 1979). Five days p.i., the infected cells were harvested, andODVs were purified from viral occlusion bodies as described previously(Braunagel et al., 1994). The final ODV pellet was resuspended in 0.5 mlof 0.1×TE (10 mM Tris, 1 mM EDTA, pH=7.5).

The purified BV and ODV virions were fractionated into envelope andnucleocapsid fractions as described previously (Braunagel et al., 1994).Final fractions were processed for SDS-PAGE and immunoblotted againsteither mouse monoclonal anti-Flag antibody (Stratagene), rabbitpolyclonal anti-VP39 antiserum (kindly provided by Lorena Passarelli,Kansas State University, USA), rabbit polyclonal anti-GP64 antiserum(kindly provided by Hualin Wang and Feifei Yin, Wuhan Institute ofVirology, China (Yin et al., 2008)), or rabbit polyclonal antiserumagainst per os infectivity factor 1 (PIF-1) (kindly provided by Ke Peng,Wageningen University, The Netherlands (Peng et al., 2010)).

Development of Transgenic Sf9-Derived Cell Line Expressing Vp80

To develop a cell line, which produces the VP80 protein, a 2105-bpfragment carrying the vp80 ORF was amplified using primers vp80-SacI-Fand vp80-XbaI-R (Table 1) and cloned into pJet1.2/Blunt, to generatepJet1.2-vp80. In the next step, the vp80 ORF was cut out (SacI/XbaI)from pJet1.2-vp80, and subcloned into SacI/XbaI-digested pIZ(Invitrogen), to create pIZ-vp80. The resulting plasmid vector pIZ-vp80was linearized with Eco57I, and gel-purified. Sf9 cells were seeded in asix-well plate (1×10⁶ cells/well), and transfected with 10 μg of thelinearized vector. After 24 hours post-transfection, cells were selectedby cell culture medium containing Zeocin™ (300 μg/ml) for 2 to 3 weeks,until no control Sf9 cells survived under the same conditions. Cellswere then propagated as an uncloned cell line.

Recombinant Protein Expression with the vp80null Virus

To measure the capacity to express recombinant protein with the Ac-Δvp80(trans-complemented) virus seed, 3.0×10⁷ non-transformed Sf9 cells wereinfected (independent triplicate assay) with Ac-wt, Ac-Δvp80-Flag.vp80(both produced in non-transformed cell line) or Ac-Δvp80 virus (producedin the Sf9-vp80 cell line) at a MOI=10. All of these virus seeds areexpressing egfp as a model heterologous gene from the baculovirus verylate p10 promoter. At 48 h and 72 h p.i. cells and culture medium wereharvested and used for Western blotting, enzyme-linked immunosorbentassay (ELISA) or BV titration (see above). For Western blotting the sameantibodies as mentioned above were used to detect the Flag-tag, EGFP,and GP64, as well as a monoclonal mouse anti-actin antibody (ImmunO).

For relative quantification, Maxisorp 96-well plates (Nunc) were coatedovernight at 4° C. with 100 ng of rabbit polyclonal anti-GFP antibody(Molecular Probes) in a volume of 100 μl per well, which was followed bystandard ELISA procedures as previously described (Fric et al., 2008).The percentage of EGFP production was calculated (independent triplicateassay) according to the formula: % EGFP expression=(testabsorbance_(nh)−background absorbance)/(Ac-wt EGFP_(72h)−backgroundabsorbance)×100%, where nh represents the time point p.i. Thestatistical significance of the observed differences between the controlAc-wt and the experimental Ac-Δvp80-Flag.vp80 and Ac-Δvp80 genotypes wasanalyzed with the Student's t-test.

Results The AcMNPV Vp80 Gene is Essential for Viral Replication

An AcMNPV deletion virus was constructed as detailed in FIG. 2. Repairconstructs were designed such that the wild-type vp80 ORF or N- andC-terminally FLAG-tagged vp80 genes along with its native or polyhedrinpromoter regions were inserted into the polyhedrin locus along with theegfp gene under the p10 promoter (FIG. 3A). To investigate the functionof the vp80 gene, Sf9 cells were transfected with either the knock-outor repair bacmid constructs and monitored for EGFP expression byfluorescence microscopy. When Ac-vp80 null was introduced into Sf9cells, no viral propagation was observed in cell culture at 72 h to 120h p.t. We could observe only a “single-cell infection” phenotype similarto the phenotype of Ac-gp64null bacmid (FIG. 3B). The results indicatethat Ac-vp80null is able to reach the very late phase of infection asconfirmed by p10 promoter-driven EGFP expression. From 72 h to 120 hoursp.t., widespread EGFP expression could be seen in insect cell monolayersthat were transfected with the three repair (vp80 driven from its nativepromoter, vp80 driven from polyhedrin promoter and N-terminallyFLAG-tagged vp80 driven from its native promoter) constructs indicatingthat these bacmids were able to produce levels of infectious buddedvirions sufficient to initiate secondary infection at a similar level asthe wild-type bacmid (FIG. 3B). In contrast, in insect cells transfectedwith C-terminally FLAG-tagged vp80 repair constructs, by 72 h p.t. EGFPexpression was only observed in isolated cells that were initiallytransfected indicating that this bacmid construct is defective in viralreplication (FIG. 3B). However, by 96 h p.t. formation of tiny plaqueswas observed and by 120 h p.t. very few plaques of normal size weredeveloped. The results show that the C-terminally flagged mutant isstrongly delayed in producing budded virus and showed that an unmodifiedC-terminus is very important for the function of VP80. At 5 days p.t.,cell culture supernatants were removed and added to freshly plated Sf9cells and then incubated for 3 days to detect infection by virusgenerated from cells transfected with these bacmids. As expected, Sf9cells incubated with supernatants from the transfections with repairconstructs showed numerous EGFP expressing cells (FIG. 3C).Nevertheless, cells incubated with supernatants from C-terminallyFLAG-tagged constructs showed a significant reduction in the number ofEGFP-positive cells. On the other hand, in insect cells incubated withsupernatants from the transfection with the vp80 knockout, no EGFPexpression was detected at any time-point analyzed up to 72 h (FIG. 3C).

Moreover, to characterize the exact effect of deletion of the vp80 geneon AcMNPV infection, the viral propagation in transfected Sf9 cells wascompared between Ac-wt, Ac-Δvp80, Ac-Δvp80-vp80Rep,Ac-Δvp80-polh-vp80Rep, Ac-Δvp80-FLAG-vp80Rep and Ac-Δvp80-vp80-FLAGRep.Cell culture supernatants of all the above bacmid constructs wereanalysed at indicated time points for BV production (FIG. 4). Asexpected, the repaired Ac-Δvp80-vp80Rep, Ac-Δvp80-polh-vp80Rep, andAc-Δvp80-FLAG-vp80Rep viruses showed kinetics of viral replicationconsistent with wild-type virus (Ac-wt) propagation. Budded virionproduction by the C-terminally flagged Ac-Δvp80-vp80-FLAGRep virus wasreduced to approximately 0.06% compared to the Ac-wt virus or the otherrepaired viruses.

These results indicate that the vp80 gene is essential for infectious BVproduction. It has clearly been proven that the whole sequence of vp80ORF can completely be deleted from the bacmid backbone and adequatelyrescued by introduction of the vp80 ORF into a heterologous site(polyhedrin locus) of the genome. We also showed that vp80 geneexpression can be driven by the heterologous polyhedrin promotersequence with no negative effect on viral replication in cell culture.Additionally, we observed that the N-terminus in contrast to theC-terminus of VP80 is permissive to gene modifications (epitopetag-labeling). We noted that the kinetics of the C-terminallyFLAG-tagged VP80 virus were significantly delayed when compared with allother rescue or wild-type viruses, indicating the functional importanceof the VP80 C-terminus.

VP80 is Required for Production of Both BV and ODV

The results described above indicated that the Ac-vp80null mutant iscompletely defective in production of infectious budded virus. However,there was also a possibility that the mutant can still producenon-infectious budded particles. To investigate the ability, Sf9 cellswere transfected with either the knock-out, repair or wild-type bacmidconstructs and 7 days p.t. cell culture mediums were ultracentrifuged topellet budded viruses. The formed pellets were either analyzed bynegative staining electron microscopy or by Western blot- and PCR-baseddetection to confirm the presence of the budded viruses. No intactbudded virus, virus-like particles, nor its structures (such as majorcapsid protein VP39 and viral genome sequence) were revealed in thepellet from the cells transfected with the Ac-vp80null mutant (FIGS. 5Aand 5B). On the other hand, all analyzed repair constructs producednormally-appearing budded virus as compared with budded virus-derivedfrom the wild-type virus (FIG. 5A). Nevertheless, it was very difficultto find representative budded virions in the pellet derived fromC-terminally FLAG-tagged vp80 gene repair construct-transfected cells.

To further characterize deletion of the vp80 gene on baculovirus lifecycle, electron microscopy was performed with ultra-thin sectionsgenerated from bacmid-transfected cells. The Ac-vp80null-transfectedcells developed typical phenotypes of baculovirus-infected cells with anenlarged nucleus, a fragmented host chromatin, an electron-densevirogenic stroma, etc. (FIG. 6A). The absence of VP80 did not preventformation of normally-appearing nucleocapsids inside the virogenicstroma (FIG. 6C). The formed nucleocapsids were phenotypicallyundistinguishable from those produced by either the Ac-vp80null repairor Ac-wt bacmids. However, an abundance of assembled nucleocapsids wasrather less as compared with cells transfected with the Ac-vp80nullrepair or Ac-wt bacmids (FIGS. 6E and 6G). In addition, noocclusion-derived virions nor bundles of nucleocapsids prior to anenvelopment could be observed in the peristromal compartment of anucleoplasm (so called the ring zone) of Ac-vp80null bacmid-transfectedcells (FIGS. 6B and 6D). It seems that VP80 plays a role duringmaturation of nucleocapsids and/or their release/transport from thevirogenic stroma. Eventually, VP80 can somehow contribute to anefficient nucleocapsid assembly which could be explained by the smallnumber of nucleocapsids present in the virogenic stroma of Ac-vp80nulltransfected cells. When the vp80 gene was re-introduced back into thebacmid mutant, a lot of nucleocapsids and occlusion-derived virionscould be seen in the ring zones of transfected cells (FIG. 6F). Anabundance and morphology of occlusion-derived virions produced inAc-Δvp80-vp80 repair bacmid-transfected cells were similar to thoseproduced by wild-type bacmid (FIGS. 6F and 6H).

VP80 is Associated with Nucleocapsids of Both BV and ODV

To investigate the association of VP80 with BV preparations, BVs werecollected at 48 h p. i. and nucleocapsid and envelope fractions wereseparated. The Flag.VP80 protein was only detected in the nucleocapsidfraction as a double-band of molecular masses ranging between 80-kDa and95-kDa that were observed in infected Sf9 cells (FIG. 14A, upper panel).Correct separation into nucleocapsid and envelope fractions wasconfirmed with antibodies against VP39 (nucleocapsid only) and GP64(envelope only) (FIG. 14A, lower panels).

To examine whether VP80 is also associated with ODVs, Sf9 cells wereco-infected with the Ac-Δvp80-Flag.vp80 and occlusion body(OB)-producing wt AcMNPV viruses to provide the POLH protein. Westernblot analysis showed that VP80 associates with the nucleocapsid fractionof ODVs and in this case migrates as a single band of ˜80 kDa,corresponding to the 80-kDa form produced in the very late phase ofinfection (FIG. 14B, upper panel). Proper fractionation intonucleocapsid and envelope fractions was controlled with antiserumagainst PIF-1, an ODV envelope protein (FIG. 14B, lower panel).

The Function of VP80 can be Rescued by Genetic Trans-Complementation

To verify whether a vp80 deletion in the viral genome can becomplemented by a vp80 ORF offered in trans under control of aconstitutive promoter, a transgenic cell line expressing Flag-taggedvp80 was constructed. In these cells VP80 was mainly produced as aprotein of approximately 95-kDa as was shown by Western blot analysiswith anti-Flag antibody (FIG. 15A). Two minor bands, one of ˜80-kDa anda second of ˜65-kDa were also observed.

In trans-complementation assays, Sf9-vp80 cells were transfected withthe Ac-Δvp80 bacmid, and the spread of virus infection was monitored byEGFP-specific fluorescence at 96 h and 120 h p.t. (FIG. 15Ba-c). Viralplaques were seen in the transfected Sf9-vp80 cells demonstrating thatthe virus was transmitted from cell to cell. Nevertheless, we noted thatthe number and size of the developed plaques was significantly smallerthan observed in Sf9 cells transfected with the Ac-wt bacmid (FIG. 15d). As a control, non-transgenic Sf9 cells showed only single-cellinfections when transfected with the Ac-Δvp80 bacmid (FIG. 15Bc).

When the culture medium of the Ac-Δvp80 transfected Sf9-vp80 cells wasused to infect freshly seeded non-transgenic Sf9 cells a “single-cellinfection” phenotype was observed (FIG. 15Bb, right panel). Hence, theBV particles resulting from trans-complementation were able to entercells but were defective in producing new BV. This also shows that theAc-Δvp80 did not revert to Ac-wt in the Sf9-vp80 cells, by picking upthe transgene from the host cells. As predicted, no EGFP signal wasdetected in Sf9 cells receiving the supernatant fromAc-Δvp80-transfected, non-transgenic Sf9 cells (FIG. 15Bc, right panel).The numbers of infectious BVs released from the Sf9-vp80 cellstransfected with the Ac-Δvp80 bacmid were compared with those producedin Sf9 cells transfected with Ac-wt at 6 days p.i. This experimentshowed that the current trans-complementation system is approximately 25fold less effective in BV production than the classical Sf9-basedproduction system (FIG. 15C).

Trans-Complemented, Replication-Deficient Ac-vp80null Virus is Competentto Express High Levels of Recombinant Protein

To assess the effect of the vp80 gene deletion on the level ofrecombinant protein expression, a bench-scale comparative productionassay has been performed. Herein, the Sf9 cells were in parallelinfected with three types of baculovirus seeds at an MOI=10, namely (i)Ac-wt, (ii) Ac-Δvp80-Flag.vp80 (both produced in Sf9 cells), and (iii)Ac-Δvp80 (produced in Sf9-vp80 cells) all encoding EGFP. Westernblotting profiles showed that the EGFP protein was expressed atidentical levels for all three tested baculovirus genotypes as was theGP64 glycoprotein which served here for control purposes (FIG. 16A,upper panel). The relative amount of EGFP was quantified by ELISA at 48and 72 h p.i. in infected cell lysates (FIG. 16B) and did not reveal anystatistically significant difference in EGFP levels between the threetested baculovirus genotypes. The results thus demonstrate that thetrans-complemented Ac-Δvp80 virus seed, although defective in viralreplication, is as capable to produce recombinant protein asconventional baculovirus expression vectors as long as the initialmultiplicity of infection is high enough to infect all cells.

Also during the production culture, revertant virus genotypes carryingthe vp80 gene were not detected, as no de novo expressed Flag.VP80protein (FIG. 16A) was detected in immunoblots. Theoretically, a certainquantity of Flag.VP80 protein associated with the trans-complementedvirus seed is entering the insect cells, but this was no longer detectedat very late times post-infection and is probably degraded by eitherlysosome- or proteasome-mediated activity. In the same experiment, no BVrelease was recorded in cell culture supernatants originated from Sf9cells inoculated with the Ac-Δvp80 virus seed (FIG. 16C), demonstratingthat neither revertant virus generation nor wild-type viruscontamination had occurred.

Summary

In this study we focused on the improvement of conventionalbaculovirus-based expression tools with the goal to eliminatecontaminating baculovirus progeny from manufactured recombinantprotein(s). This effort is strongly driven by pharmaceuticalperspectives, since recombinant baculovirus-expressed therapeutics arebeing more and more used in human and veterinary medicine. Hence, weaimed to identify baculovirus gene(s) whose targeting results in adeficiency of baculovirus virion production, but does not or only mildlyaffects very late gene expression. In this way high level expression ofheterologous genes will be safeguarded.

A summarizing overview of the new technology with the vp80 gene asexample is presented in FIG. 16. Using bacmid-based engineering theinventors constructed an AcMNPV genome lacking the vp80 gene (FIG. 16B).Functional genomics and electron microscopy analyses revealed that vp80deficiency prevents production of both BVs and ODVs. In parallel, Sf9cells were engineered to produce VP80 to trans-complement the Ac-Δvp80knock-out bacmid (FIG. 14A,C). Finally, we proved thattrans-complemented, replication-deficient baculovirus seed is capable ofproducing an amount of recombinant protein similar to that produced byconventional baculovirus vectors (FIG. 14D).

TABLE 1 List of PCR primers in order of appearance in the text. SEQ IDOrien- # Primer name Sequence tation  2 vp39-F5′-gcttctaatacgactcactatagggtcgtatccgctaagcgttct-3′ Forward  3 vp39-R5′-gcttctaatacgactcactatagggacgcaacgcgttatacacag-3′ Reverse  4 455105′-gcttctaatacgactcactatagggacagcgtgtacgagtgcat-′3 Forward  5 462355′-gcttctaatacgactcactatagggatctcgagcgtgtagctggt-3′ Reverse  6 902925′-gcttctaatacgactcactatagggtaccgccgaacattacacc-3′ Forward  7 908895′-gcttctaatacgactcactatagggtctattggcacgtttgct-3′ Reverse  8 ec-27-F5′-gcttctaatacgactcactatagggaaagcagacactcggcagat-3′ Forward  9 ec-27-R5′-gcttctaatacgactcactatagggttgagtggcttcaacctcag-3′ Reverse 10 dbp-F5′-gcttctaatacgactcactatagggcgctcgctagttttgttct-3′ Forward 11 dbp-R5′-gcttctaatacgactcactatagggaaagatcggaaggtggtga-3′ Reverse 12 gfp-F5′-gcttctaatacgactcactatagggctgaccctgaagttcatctg-3′ Forward 13 gfp-R5′-gcttctaatacgactcactatagggaactccagcaggaccatgt-3′ Reverse 14 cat-F5′-gcttctaatacgactcactatagggacggcatgatgaacctgaat-3′ Forward 15 cat-R5′-gcttctaatacgactcactatagggatcccaatggcatcgtaaag-3′ Reverse 16 vp80-ko-F5′-ctgtattgtaatctgtaagcgcacatggtgcattcgatataaccttataatgtgt- Forwardgctggaatgccct-3′ 17 vp80-ko-R5′-aaatgtactgaatataaataaaaattaaaaatattttataattttttatttaccgtt- Reversecgtatagcatacat-3′ 18 89507 5′-agcggtcgtaaatgttaaacc-3′ Forward 19 917135′-tgtataaacaatatgttaatatgtg-3′ Reverse 20 gfp-NheI-F5′-ccaaaccgctagcaacatggtgagcaagggcgag-3′ Forward 21 gfp-SphI5′-aggaaagggcatgcttaacgcgtaccggtcttgtacagctcgtccatgc-3′ Reverse 22pvp80-StuI-F 5′-ggaacaaaggcctgagctcaaagtaagacctttactgtcc-3′ Forward 23vp80-XbaI-R 5′-ccttctatctagattatataacattgtagtttgcg-3′ Reverse 24vp80-SacI-F 5′-ttatcttgagctcaatatgaacgattccaattctc-3′ Forward 25vp80-FLAG-R1 5′-caacagagaattggaatcgttcttatcgtcgtcatccttgtaatc- Reversecatattataaggttatatcgaatg-3′ 26 vp80-FLAG-R5′-ccttctatctagattacttatcgtcgtcatccttgtaatctataacat- Reversetgtagtttgcgttc-3′ 27 M13-F 5′-cccagtcacgacgttgtaaaacg-3′ Forward 28M13-R 5′-agcggataacaatttcacacagg-3′ Reverse 29 GenR5′-agccacctactcccaacatc-3′ Reverse 30 vp39-ko-F5′-cttcttatcgggttgtacaac-3′ Forward 31 vp39-ko-R5′-gcgtatcatgacgatggatg-3′ Reverse 32 vp39-SacI-F5′-aaggttctctagattagacggctattcctccac-3′ Forward 33 vp39-XbaI-R5′-ttatcttgagctcaatatggcgctagtgcccg-3′ Reverse 34 vp39-StuI-F5′-ggaacaaaggcctgagctcttagacggctattcctccac-3′ Forward 35 lef-4-XbaI-R5′-ccttctatctagattaatttggcacgattcggtc-3′ Reverse 36 cg-30-XbaI-F5′-aaggttctctagattaatctacatttattgtaacatttg-3′ Forward 37 vp39-FLAG-SacI-5′-ttatcttgagctcaatatggattacaaggatgacgacgataaggc- Reverse Rgctagtgcccgtgggt-3′ 38 vp1054-ko-F5′-gtactgaaagataatttatttttgatagataataattacattattttaa- Forwardacgtgttcgaccaagaaaccgat-3′ 39 vp1054-ko-R15′-agggcgaattccagcacactttattacgtggacgcgttactttgc-3′ Reverse 40vp1054-ko-R2 5′-gataagaatgcttgtttaacaaataggtcagctgttaaatact- Reverseggcgatgtaccgttcgtatagcatacat-3′ 41 vp1054-Rep-F5′-ggttgtttaggcctgagctcctttggtacgtgttagagtgt-3′ Forward 42 vp1054-Rep-R5′-tcctttcctctagattacacgttgtgtgcgtgcaga-3′ Reverse 43 p6.9-ko-F5′-gcttcgttcattcgctactgtcggctgtgtggaatgtctggttgtt- Forwardaagtgtgctggaattcgccct-3′ 44 p6.9-ko-R5′-aatattaataaggtaaaaattacagctacataaattacacaattta- Reverseaactaccgttcgtatagcatacat-3′ 45 Ac-p6.9-F5′-tttgaattcatggttgcccgaagctccaagac-3′ Forward 46 Ac-p6.9-R5′-tttgcggccgcttaatagtagcgtgttctgtaac-3′ Reverse 47 Se-p6.9-F5′-tttgaattcatgtatcgtcgtcgttcatc-3′ Forward 48 Se-p6.9-R5′-tttgcggccgcttaatagtggcgacgtctgtatc-3′ Reverse 49 865965′-gggcttagtttaaaatcttgca-3′ Forward 50 869955′-aattcaaacgaccaagacgag-3′ Reverse 51 45122 5′-gcaatcatgacgaacgtatgg-3′Forward 52 46441 5′-cgataatttttccaagcgctac-3′ Reverse 53 pp6.9-F5′-ggtcgacgtaccaaattccgttttgcgacg-3′ Forward 54 pp6.9-R5′-ggtcgacggatccgtttaaattgtgtaatttatg-3′ Reverse 55 758345′-cttcttatcgggttgtacaac-3′ Forward 56 76420 5′-gcgtatcatgacgatggatg-3′Reverse

REFERENCES

-   Abe, T., Takahashi, H., Hamazaki, H., Miyano-Kurosaki, N.,    Matsuura, Y. & Takaku, H. (2003). Baculovirus induces an innate    immune response and confers protection from lethal influenza virus    infection in mice. Journal of Immunology 171, 1133-1139.-   Aslanidi, G., Lamb, K. & Zolotukhin, S. (2009). An inducible system    for highly efficient production of recombinant adeno-associated    virus (rAAV) vectors in insect Sf9 cells. Proceedings of the    National Academy of Sciences USA 106, 5059-5064.-   Boyce, F. M. & Bucher, N. L. R. (1996). Baculovirus-mediated gene    transfer into mammalian cells. Proceedings of the National Academy    USA 93, 2348-2352.-   Braunagel, S. C. & Summers, M. D. Autographa californica nuclear    polyhedrosis virus, PDV, and ECV viral envelopes and nucleocapsids:    Structural proteins, antigens, lipid and fatty acid profiles.    Virology 202, 315 (1994).-   Bright, R. A., Carter, D. M., Crevar, C. J., Toapanta, F. R.,    Steckbeck, J. D., Cole, K. S., Kumar, N. M., Pushko, P., Smith, G.,    Tumpey, T. M. & Ross, T. M. (2008). Cross-clade protective immune    responses to influenza viruses with H5N1 HA and NA elicited by an    influenza virus-like particle. PLoS ONE 3.-   Carbonell, L. F., Klowden, M. J. & Miller, L. K. (1985).    Baculovirus-mediated expression of bacterial genes in dipteran and    mammalian cells. Journal of Virology 56, 153-160.-   Carbonell L. F., Hodge M. R., Tomalski, M. D., Miller, L. K. (1988).    Synthesis of a gene coding for an insect-specific scorpion    neurotoxin and attempts to express it using baculovirus vectors.    Gene 73, 409-18.-   Charlton, C. A. & Volkman, L. E. (1991). Sequential rearrangement    and nuclear polymerization of actin in baculovirus-infected    Spodoptera frugiperda cells. Journal of Virology 65, 1219-27.-   Charlton, C. A. & Volkman, L. E. (1993). Penetration of Autographa    californica nuclear polyhedrosis virus nucleocapsids into IPLB Sf 21    cells induces actin cable formation. Virology 197, 245-54.-   Cohen, D. P. A., Marek, M., Davies, B. G., Vlak, J. M. & van    Oers, M. M. (2009). Encyclopedia of Autographa californica    nucleopolyhedrovirus genes. Virologica Sinica 24, 359.-   Condreay, J. P.& Kost, T. A. (2007). Baculovirus expression vectors    for insect and mammalian cells. Curr Drug Targets 8, 1126-31.-   Cox, M. M. J. & Hollister, J. (2009). FluBlok, A next generation    influenza vaccine manufactured in insect cells. Biologicals 37,    182-189.-   Dai, X., Willis, L. G., Palli, S. R. & Theilmann, D. A. (2005).    Tight transcriptional regulation of foreign genes in insect cells    using an ecdysone receptor-based inducible system. Protein    Expression and Purification 42, 236-245.-   Datsenko, K. A. & Wanner, B. L. (2000). One-step inactivation of    chromosomal genes in Escherichia coli K-12 using PCR products.    Proceedings of the National Academy of Sciences USA 97, 6640-6645.-   Fric, J., Marek, M., Hrusková, V., Holán, V. & Forstová J. (2008).    Cellular and humoral immune responses to chimeric EGFP-pseudocapsids    derived from the mouse polyomavirus after their intranasal    administration. Vaccine 26, 3242.-   Friesen. P. D. & Miller, L. K. (1986). The regulation of baculovirus    gene expression in: “The Molecular Biology of Baculoviruses” (W.    Doerfler and P. Boehm, eds.) Springer-Verlag, Berlin, pp. 31-49.-   Funk, C. J. & Consigli, R. A. (1993). Phosphate cycling on the basic    protein of Plodia interpunctella granulosis virus. Virology 193,    396-402.-   Gheysen, D., Jacobs, E., De Foresta, F., Thiriart, C., Francotte,    M., Thines, D. & De Wilde, M. (1989). Assembly and release of HIV-1    precursor pr55(gag) virus-like particles from recombinant    baculovirus-infected insect cells. Cell 59, 103-112.-   Gronowski, A. M., Hilbert, D. M., Sheehan, K. C. F., Garotta, G. &    Schreiber, R. D. (1999). Baculovirus stimulates antiviral effects in    mammalian cells. Journal of Virology 73, 9944-9951.-   Harper, D. M., Franco, E. L., Wheeler, C. M., Moscicki, A.-B.,    Romanowski, B., Roteli-Martins, C. M., Jenkins, D., Schuind, A.,    Costa Clemens, S. A. & Dubin, G. (2006). Sustained efficacy up to    4.5 years of a bivalent L1 virus-like particle vaccine against human    papillomavirus types 16 and 18: follow-up from a randomised control    trial. The Lancet 367, 1247-1255.-   Hill-Perkins, M. S., &, Possee, R. D. (1990). A baculovirus    expression vector derived from the basic protein promoter of    Autographa californica nuclear polyhedrosis virus. Journal of    General Virology 71: 971-976.-   Hofmann, C., Sandig, V., Jennings, G., Rudolph, M., Schlag, P. &    Strauss, M. (1995). Efficient gene transfer into human hepatocytes    by baculovirus vectors. Proceedings of the National Academy of    Sciences USA 92, 10099-10103.-   Jeong, S. H., Qiao, M., Nascimbeni, M., Hu, Z., Rehermann, B.,    Murthy, K. & Liang, T. J. (2004). Immunization with hepatitis C    virus-like particles induces humoral and cellular immune responses    in nonhuman primates. Journal of Virology 78, 6995-7003.-   Jing Chen, S., Er Hui, Z., Lun Guang, Y., Hong Ling, Z. & Peng    Fei, J. (2009). A high efficient method of constructing recombinant    Bombyx mori (silkworm) multiple nucleopolyhedrovirus based on    zero-background Tn7-mediated transposition in Escherichia coli.    Biotechnology Progress 25, 524-529.-   Kaikkonen, M. U., Viholainen, J. I., Narvanen, A., Yla-Herttuala, S.    & Airenne, K. J. (2008). Targeting and purification of metabolically    biotinylated baculovirus. Human Gene Therapy 19, 589-600.-   Kanginakudru, S. S., Royer C., Edupalli S. V., Jalabert A., Mauchamp    B., Chandrashekaraiah, Prasad S. V., Chavancy G., Couble P.,    Nagaraju J. (2007). Targeting ie-1 gene by RNAi induces baculoviral    resistance in lepidopteran cell lines and in transgenic silkworms.    Insect molecular biology 16, 635-644.-   Kato, T., Kajikawa, M., Maenaka, K. & Park, E. Y. (2010). Silkworm    expression system as a platform technology in life science. Applied    Microbiology and Biotechnology 85, 459-470.-   Kelly, D. C., Brown, D. A., Ayres, M. D., Allen, C. J. &    Walker, I. O. (1983). Properties of the major nucleocapsid protein    of Heliothis zea singly enveloped nuclear polyhedrosis virus.    Journal of General Virology 64, 399-408.-   Kitajima, M. & Takaku, H. (2008). Induction of antitumor acquired    immunity by baculovirus Autographa californica multiple nuclear    polyhedrosis virus infection in mice. Clinical and Vaccine    Immunology 15, 376-378.-   Kost, T. A., Condreay, J. P. & Jarvis, D. L. (2005). Baculovirus as    versatile vectors for protein expression in insect and mammalian    cells. Nature Biotechnology 23, 567-575.-   Lackner, A., Genta, K., Koppensteiner, H., Herbacek, I., Holzmann,    K., Spiegl-Kreinecker, S., Berger, W. & Grusch, M. (2008). A    bicistronic baculovirus vector for transient and stable protein    expression in mammalian cells. Analytical Biochemistry 380, 146-148.-   Lebacq-Verheyden A M, Kasprzyk P G, Raum M G, Van Wyke Coelingh K,    Lebacq J A, Battey J F. (1988) Posttranslational processing of    endogenous and of baculovirus-expressed human gastrin-releasing    peptide precursor. Molecular and Cell Biology 8, 3129-35.-   Lesch, H. P., Turpeinen, S., Niskanen, E. A., Mähönen, A. J.,    Airenne, K. J. & Ylä-Herttuala, S. (2008). Generation of lentivirus    vectors using recombinant baculoviruses. Gene Therapy 15, 1280-1286.-   Li, X., Pang, A., Lauzon, H. A. M., Sohi, S. S. & Arif, B. M.    (1997). The gene encoding the capsid protein P82 of the    Choristoneura fumiferana multicapsid nucleopolyhedrovirus:    Sequencing, transcription and characterization by immunoblot    analysis. Journal of General Virology 78, 2665-2673.-   Liu, X., Li, K., Song, J., Liang, C., Wang, X. & Chen, X. (2006a).    Efficient and stable gene expression in rabbit intervertebral disc    cells transduced with a recombinant baculovirus vector. Spine 31,    732-735.-   Liu, Y. K., Chu, C. C. & Wu, T. Y. (2006b). Baculovirus ETL promoter    acts as a shuttle promoter between insect cells and mammalian cells.    Acta Pharmacologica Sinica 27, 321-327.-   Lopez, M. G., Alfonso, V., Carrillo, E. & Taboga, 0. (2009).    Trans-complementation of polyhedrin by a stably transformed Sf9    insect cell line allows occ-baculovirus occlusion and larval per os    infectivity. Journal of Biotechnology 145, 199-205.-   Lu, A. & Carstens, E. B. (1992). Nucleotide sequence and    transcriptional analysis of the p80 gene of Autographa californica    nuclear polyhedrosis virus: a homologue of the Orgyia pseudotsugata    nuclear polyhedrosis virus capsid-associated gene. Virology 190,    201-209.-   Luckow, V. A. & Summers, M. D. (1988). Trends in the development of    baculovirus expression vectors. Bio/Technology 6, 47-55.-   Luckow, V. A., Lee, S. C., Barry, G. F. & Olins, P. O. (1993).    Efficient generation of infectious recombinant baculoviruses by    site-specific transposon-mediated insertion of foreign genes into a    baculovirus genome propagated in Escherichia coli. Journal of    Virology 67, 4566-79.-   Ludwig, C. & Wagner, R. (2007). Virus-like particles-universal    molecular toolboxes. Current Opinion in Biotechnology 18, 537-545.-   Lung, O., Westenberg, M., Vlak, J. M., Zuidema, D. & Blissard, G. W.    (2002). Pseudotyping Autographa californica multicapsid    nucleopolyhedrovirus (AcMNPV): F proteins from group II NPVs are    functionally analogous to AcMNPV GP64. Journal of Virology. 76,    5729-5736.-   Maeda, S., Kawai, T., Obinata, M., Fujiwara, H., Horiuchi, T.,    Saeki, Y., Sato, Y. & Furusawa M. (1985). Production of human    alpha-interferon in silkworm using a baculovirus vector. Nature 315,    592-4.-   Maranga, L., Rueda, P., Antonis, A. F., Vela, C., Langeveld, J. P.,    Casal, J. I, & Carrondo, M. J. (2002). Large scale production and    downstream processing of a recombinant porcine parvovirus vaccine.    Applied Microbiology Biotechnology 59, 45-50.-   Martin, B. M., Tsuji, S., LaMarca, M. E., Maysak, K., Eliason, W.,    Ginns, E. I. (1988). Glycosylation and processing of high levels of    active human glucocerebrosidase in invertebrate cells using a    baculovirus expression vector. DNA 7, 99-106.-   McKenna, K. A., Hong, H., van Nunen & Granados, R. R. (1989).    Establishment of new Trichoplusia ni cell lines in serum-free medium    for baculovirus and recombinant protein production. Journal of    Invertebrate Pathology 71, 82-90.-   Mellado, M. C., Peixoto, C., Cruz, P. E., Carrondo, M. J. &    Alves, P. M. (2008) Purification of recombinant rotavirus VP7    glycoprotein for the study of in vitro rotavirus-like particles    assembly. Journal of Chromatography B Analyicalt Technology    Biomedical Life Science. 874, 89-94.-   Miller, D. W., Safer, P. & Miller, L. K. (1986). in Genetic    Engineering: Principles and Methods Vol. 8 (eds Setlow, J. &    Hollaender, A.) Plenum Publishing, New York, pp. 277-298.-   Miller, L. K. (1988). Baculoviruses as gene expression vectors.    Annual Review Microbiology. 42, 177-99.-   Miyajima, A, Schreurs, J., Otsu, K., Kondo, A., Arai, K. & Maeda, S.    (1987). Use of the silkworm, Bombyx mori, and an insect baculovirus    vector for high-level expression and secretion of biologically    active mouse interleukin-3. Gene 58, 273-81.-   Mortola, E. & Roy, P. (2004). Efficient assembly and release of SARS    coronavirus-like particles by a heterologous expression system. FEBS    Letters 576, 174-178.-   Muller, R., Pearson, M. N., Russell, R. L. Q. & Rohrmann, G. F.    (1990). A capsid-associated protein of the multicapsid nuclear    polyhedrosis virus of Orgyia pseudotsugata: Genetic location,    sequence, transcriptional mapping, and immunocytochemical    characterization. Virology 176, 133-144.-   Murges, D., Kremer, A. & Knebel-Morsdorf, D. (1997). Baculovirus    transactivator 1E1 is functional in mammalian cells. Journal of    General Virology 78, 1507-1510.-   Noad, R. & Roy, P. (2003). Virus-like particles as immunogens.    Trends in Microbiology 11, 438-444.-   Olszewski, J. & Miller, L. K. (1997). Identification and    characterization of a baculovirus structural protein, VP1054,    required for nucleocapsid formation. Journal of Virology 71,    5040-50.-   Peng, K., van Oers, M. M., Hu, Z. H., van Lent, J. W. M.,    Vlak, J. M. (2010). Baculovirus per os infectivity factors form a    complex on the surface of occlusion derived virus. Journal of    Virology (in press).-   Pijlman, G. P., Roode, E. C., Fan, X., Roberts, L. O., Belsham, G.    J., Vlak, J. M. & van Oers, M. M. (2006). Stabilized baculovirus    vector expressing a heterologous gene and GP64 from a single    bicistronic transcript. Journal of Biotechnology 123, 13-21.-   Ramadan, N., Flockhart, I., Booker, M., Perrimon, N. &    Mathey-Prevot, B. (2007). Design and implementation of    high-throughput RNAi screens in cultured Drosophila cells. Nature    Protocols 2, 2245-2264.-   Ramqvist, T., Andreasson, K. & Dalianis, T. (2007). Vaccination,    immune and gene therapy based on virus-like particles against viral    infections and cancer. Expert Opinion on Biological Therapy 7,    997-1007.-   Salem, T. Z. & Maruniak, J. E. (2007). A universal transgene    silencing approach in baculovirus-insect cell system. Journal of    Virological Methods 145, 1-8.-   Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular    Cloning. A Laboratory Manual, Cold Spring Harbor, N.Y.-   Slack, J. & Arif, B. M. (2006). The baculoviruses occlusion-derived    virus: virion structure and function. Advances in virus research 69,    99-165.-   Smith, G. E, Ju, G., Ericson, B. L, Moschera, J., Lahm, H. W,    Chizzonite, R. & Summers, M. D. (1985). Modification and secretion    of human interleukin 2 produced in insect cells by a baculovirus    expression vector. Proceedings National Academy of Sciences USA 82,    8404-8.-   Smith, R. H, Levy, J. R. & Kotin, R. M. (2009). A simplified    baculovirus-AAV expression vector system coupled with one-step    affinity purification yields high-titer rAAV stocks from insect    cells. Molecular Therapy 17, 1888-1896.-   Smith, G. E. & Summers, M. D. (1979). Restriction maps of five    Autographa californica MNPV variants, Trichoplusia ni MNPV, and    Galleria mellonella MNPV DNAs with endonucleases SmaI, KpnI, BamHI,    SacI, XhoI, and EcoRI. Journal of Virology 30, 828-838.-   Suzuki, N., Nonaka, H., Tsuge, Y., Okayama, S., Inui, M. &    Yukawa, H. (2005). Multiple large segment deletion method for    Corynebacterium glutamicum. Applied Microbiology and Biotechnology    69, 151-161.-   Tang, X.-D., Xu, Y.-P., Yu, L.-I., Lang, G.-J., Tian, C.-H., Zhao,    J.-F. & Zhang, C.-X. (2008). Characterization of a Bombyx mori    nucleopolyhedrovirus with Bmvp80 disruption. Virus Research 138,    81-88.-   Tellez, M. (2005). Process optimization protocol for tangential flow    filtration of insect cells and baculovirus. Presented at WilBio    Conference on Baculovirus & Insect Cell Culture—Process Development    and Production Issues, Savannah/Georgia, 21^(st)-24^(th) February,    2005.-   Thiem, S. M. & Miller, L. K. (1989a). A baculovirus gene with a    novel transcription pattern encodes a polypeptide with a zinc finger    and a leucine zipper. Journal of Virology 63, 4489-4497.-   Thiem, S. M. & Miller, L. K. (1989b). A baculovirus gene with a    novel transcription pattern encodes a polypeptide with a zinc finger    and a leucine zipper. Journal of Virology 63, 4489-97.-   Thiem, S. M. & Miller, L. K. (1989c). Identification, sequence, and    transcriptional mapping of the major capsid protein gene of the    baculovirus Autographa californica nuclear polyhedrosis virus.    Journal of Virology 63, 2008-2018.-   Tjia, S. T., Meyer zu Altenschildesche, G. & Doerfler, W. (1983).    Autographa californica nuclear polyhedrosis virus (AcNPV) DNA does    not persist in mass cultures of mammalian cells. Virology 125,    107-117.-   Urabe, M., Ding, C. & Kotin, R. M. (2002). Insect cells as a factory    to produce adeno-associated virus type 2 vectors. Human Gene Therapy    13, 1935-1943.-   van Lent, J. W. M., Groenen, J. T. M., Klinge-Roode, E. C.,    Rohrmann, G. F., Zuidema, D. & Vlak, J. M. (1990). Localization of    the 34 kDa polyhedron envelope protein in Spodoptera frugiperda    cells infected with Autographa california nuclear polyhedrosis    virus. Archives of Virology 111, 103-114.-   van Oers, M. M. (2006). Vaccines for Viral and Parasitic Diseases    Produced with Baculovirus Vectors. In Advances in Virus Research 68.    193-253.-   Vlak J. M., Klinkenberg, F. A, Zaal, K. J., Usmany, M.,    Klinge-Roode, E. C., Geervliet, J. B, Roosien. J, & van Lent, J. W.    (1988). Functional studies on the p10 gene of Autographa californica    nuclear polyhedrosis virus using a recombinant expressing a    p10-beta-galactosidase fusion gene. Journal of General Virology 69,    765-76.-   Wang, M. Y, Kuo, Y. Y., Lee, M. S, Doong, S. R, Ho, J. Y, Lee, L. H.    (2000). Self-assembly of the infectious bursal disease virus capsid    protein, rVP2, expressed in insect cells and purification of    immunogenic chimeric rVP2H particles by immobilized metal-ion    affinity chromatography. Biotechnology and Bioeneneering 67, 104-11.-   Wu, W., Liang, H., Kan, J., Liu, C., Yuan, M., Liang, C., Yang, K. &    Pang, Y. (2008). Autographa californica multiple    nucleopolyhedrovirus 38K is a novel nucleocapsid protein that    interacts with VP1054, VP39, VP80, and itself. Journal of Virology    82, 12356-12364.-   Yin, F., M. Wang, Y. Tan, F. Deng, J. M. Vlak, Z. Hu, and H.    Wang. 2008. A functional F analogue of AcMNPV GP64 from the Agrotis    segetum granulovirus. Journal of Virology 82, 8922-8926.

1. (canceled)
 2. A method for the production of a biopharmaceuticalproduct, comprising: (a) infecting a biopharmaceutical-producing insectcell with at least one baculovirus, said at least one baculoviruscomprising a genome coding for said biopharmaceutical product, and (b)maintaining the biopharmaceutical-producing insect cell under conditionssuch that the biopharmaceutical product is produced, wherein the genomeof said at least one baculovirus is deficient for the p6.9 gene orwherein said biopharmaceutical-producing insect cell comprises anexpression control system allowing the inactivation of the p6.9 gene. 3.The method according to claim 2, wherein the p6.9 gene is made deficientin said genome by way of nucleotide substitution, insertion or deletion.4. The method according to claim 2, wherein thebiopharmaceutical-producing insect cell is a recombinant insect cellcomprising a construct expressing a dsRNA specific for the p6.9 gene,the dsRNA being optionally expressed under an inducible promoter.
 5. Themethod according to claim 2, wherein the at least one baculovirus isproduced before step (a) in a baculovirus-producing cell expressing acomplementing copy of the p6.9 gene.
 6. The method according to claim 2,wherein the genome of said at least one baculovirus is further deficientfor at least one gene selected from vp80, vp1054 and vp39 or whereinsaid biopharmaceutical-producing insect cell further comprises anexpression control system allowing the inactivation of at least one geneselected from vp80, vp1054 and vp39.
 7. The method according to claim 2,wherein the deficiency or inactivation of the p6.9 gene does not affectvery late expression from said baculovirus in comparison to very lateexpression from wild-type baculovirus.
 8. The method according to claim2, wherein the at least one baculovirus is derived from AcMNPV or BmNPV.9. The method according to claim 2, wherein the biopharmaceuticalproduct is a recombinant protein, a recombinant virus or a virus-likeparticle.
 10. The method according to claim 9, wherein thebiopharmaceutical product is a recombinant AAV.
 11. The method accordingto claim 2, wherein the biopharmaceutical product is coded by at leastone gene introduced in the recombinant baculovirus genome under thecontrol of the polyhedrin or p10 promoter.
 12. A bacmid comprising abaculoviral genome, wherein said genome is deficient for the p6.9 gene.13. The bacmid according to claim 12, wherein said genome is furtherdeficient for at least one gene selected from vp80, vp1054 and vp39. 14.The bacmid according to claim 12, wherein said genome is derived fromAcMNPV.
 15. A recombinant baculovirus vector, wherein the genome of saidbaculovirus vector is deficient for the p6.9 gene.
 16. The recombinantbaculovirus vector according to claim 15, wherein the genome of saidbaculovirus is further deficient for at least one gene selected fromvp80, vp1054 and vp39.
 17. The recombinant baculovirus vector accordingto claim 15, wherein said vector is an AcMNPV baculovirus vector.
 18. Aninsect cell infected with a recombinant baculovirus vector comprising agenome which is deficient for the p6.9 gene.
 19. The insect cellaccording to claim 18, wherein said genome is further deficient for atleast one gene selected from vp80, vp1054 and vp39.
 20. The insect cellaccording to claim 18, wherein said genome is derived from AcMNPV. 21.An insect cell, comprising a construct expressing a dsRNA specific ofthe p6.9 gene.
 22. The insect cell according to claim 21, wherein saidconstruct is integrated in the genome of the insect cell.
 23. The insectcell according to claim 21, wherein said insect cell further comprises aconstruct expressing a dsRNA specific of at least one gene selected fromvp80, vp1054 and vp39.
 24. A cell comprising an expression cassettecoding for the p6.9 gene.
 25. The cell according to claim 24, whereinsaid cell further comprises an expression cassette coding for at leastone gene selected from vp80, vp1054 and vp39.
 26. The cell according toclaim 24, which is an insect cell.
 27. A method for the production of abaculovirus deficient for the p6.9 gene, comprising the step oftransfecting an insect cell comprising an expression cassette coding forthe p6.9 gene with a bacmid comprising a baculoviral genome, whereinsaid genome is deficient for the p6.9 gene.
 28. The method according toclaim 27, wherein said insect cell further comprises an expressioncassette coding for at least one gene selected from vp80, vp1054 andvp39 and wherein said baculoviral genome is further deficient for saidat least one gene selected from vp80, vp1054 and vp39.